The Interactive Fly
Zygotically transcribed genes
miRNAs:
miRNAs are small non-coding RNA molecule (ca. 22 nucleotides) which function in transcriptional and post-transcriptional regulation of gene expression. Most miRNAs are transcribed by DNA-dependent RNA polymerase II (RNAPII) to generate a primary miRNA (pri-miRNA) containing a region of imperfect dsRNA, known as the stem-loop structure, that harbors the future mature miRNA. Primary miRNA transcripts seem largely like the transcripts of protein-coding genes. The production of conventional miRNAs from these precursors proceeds through two site-specific cleavage events (see MicroRNA biogenesis in Drosophila melanogaster). Processing likely begins with a dsRBD protein, Pasha/DiGeorge syndrome critical region gene 8 (DGCR8; Pasha in Drosophila), binding to the pri-miRNA and recruiting the RNase III enzyme Drosha to form a multiprotein complex called the Microprocessor. This complex recognizes the duplex character of the pri-miRNA. The pri-miRNA is cleaved by Drosha to liberate a ~60-70-nt precursor miRNA (pre-miRNA) from the primary transcript. The nuclear export protein Exportin 5 recognizes the 2-nt single-stranded 3′ overhang of the pre-miRNA (characteristic of RNase III-mediated cleavage) and actively transports it in a Ran-GTP-dependent manner to the cytoplasm (Czech, 2011).
Once in the cytoplasm, the pre-miRNA is cleaved into a ~22-23-nt miRNA:miRNA* duplex by Dicer. For this purpose, the sole mammalian Dicer partners with the dsRBD protein TAR RNA-binding protein 2 (TARBP2, also known as TRBP), whereas the Drosophila melanogaster miRNA-generating Dicer 1 (DCR1) similarly interacts with a specific isoform of its dsRBD protein partner Loquacious (LOQS-PB). Small RNA duplexes generated by Dicer (and its protein partner) exhibit 2-nt single-stranded 3′ overhangs at both ends, a signature of RNase III cleavage (Czech, 2011).
Several unconventional miRNAs that are defined by their use of alternative maturation strategies have now been noted. For example, mirtrons have been found in flies and mammals. Mirtrons bypass the Drosha processing step and instead use the splicing machinery to generate pre-miRNAs. Mirtrons are very short introns and are excised, debranched and refolded into short stem-loop structures that mimic pre-miRNAs and are processed into mature miRNAs by Dicer. A few recently discovered mirtrons in flies are initially generated with extended 3′ tails that must be resected by the exosome to form a pre-miRNA suitable for Dicer processing. Once produced, small RNAs and, in many cases, specific small RNA strands must be loaded into Argonaute proteins. Sorting is influenced by the Dicer that processes the precursor, the structure of the small RNA duplex, its terminal nucleotides, its thermodynamic properties and the destination AGO protein (Czech, 2011).
In mammals, a single Dicer assorts siRNAs (see below) and miRNAs among four Argonaute subfamily proteins, apparently without much discrimination. However, in Drosophila, two distinct Dicer proteins process small RNA duplexes that preferentially enter AGO1 or AGO2 complexes. Generally, AGO1 is occupied by miRNAs, whereas AGO2 associates with siRNAs. This parallels the processing of miRNAs by Dicer 1 and siRNAs by Dicer 2. siRNAs are derived from duplexes featuring perfect or nearly perfect dsRNA, whereas miRNAs originate from precursors that typically contain several mismatches or bulges. Other features that affect sorting include the terminal nucleotides and thermodynamic properties of the duplex ends (Czech, 2011).
siRNAs:
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length. siRNAs interferes with the expression of specific genes with complementary nucleotide sequence and also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome.
The process of converting dsRNA into small RNAs is perhaps currently best understood in Drosophila (see Production of small interfering RNAs). Here, the experimental introduction of long dsRNAs results in the production of exo-siRNAs that are ~21 nt in size. long dsRNAs are processed into siRNA duplexes through sequential cleavage events by the RNase III protein Dicer 2 in collaboration with its dsRBD co-factor, a particular Loquacious isoform, LOQS-PD42,70. Dicer 2 also interacts with another dsRBD protein R2D2, but only LOQS-PD enhances siRNA production. Recent studies indicate a role of R2D2 in loading siRNA duplexes into RISC, suggesting that these two dsRBD proteins may have distinct and sequential functions (Czech, 2011).
A similar situation has been described in mammals; however, the range of cell types in which dsRNAs are produced and converted into siRNAs seems to be limited. Thus far, endo-siRNAs have been detected in abundance only in mouse oocytes and embryonic stem (ES) cells. The dsRNA triggers that give rise to murine endo-siRNAs are predicted to arise from trans interactions between gene and pseudogene transcripts, from overlapping transcription units and from transcripts that can form long hairpins. As in flies, endo-siRNA biogenesis is dependent on Dicer and, presumably, its dsRBD partners. Assorting siRNAs into Ago2 is described above (Czech, 2011).
piRNAs
Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells. They are distinct from microRNA (miRNA) in size (26-31 nt rather than 21-24 nt), lack of sequence conservation, and have increased complexity.
Small RNAs are sorted to confer association with specific Argonaute family proteins, which function as the core of the RNA-induced silencing complex (RISC). Argonaute proteins can be classified into three subgroups according to their sequence relationships: the AGO subfamily, the Piwi subfamily and the worm-specific WAGO clade. Piwi subfamily proteins load small RNAs derived from single-stranded precursors (piRNAs) and AGO clade proteins usually associate with small RNA duplexes processed by RNase III endonucleases (miRNAs and siRNAs). Small RNAs that occupy WAGO clade proteins are usually direct products of RNA synthesis (Czech, 2011).
RNAi - RNAi or RNA silencing is the process whereby double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA (Nishikura, 2001).
PTGS - Posttranscriptional gene silencing - in plants and animals and 'quelling' in fungi are RNAi-like mechanisms whereby naturally produced
stem-loop RNA precursors are processed to produce interfering microRNAs that silence translation of mRNA transcripts. Repression of sequence-related viral genes occurs in plants undergoing PTGS, whereas quelling (simultaneous silencing or cosuppression) of homologous endogenous genes and transgenes has been observed in fungi. Cosuppression is functional silencing of chromosomal loci induced by transgenes (Nishikura, 2001).
siRNA - short interfering RNA - involved in silencing of selfish genetic elements such as transposons - 21-25 nt RNA fragments - siRNAs are processed from longer double-stranded (dsRNA) precursor molecules with perfect complementarity. The dsRNAs are cleaved into small interfering RNAs (siRNAs) that are 21-23 nucleotide duplexes. They act as guides for a siRNA-induced silencing complex (siRISC) to target complementary mRNAs. If such an mRNA molecule is found, the base pairing interactions between siRNA and complementary sites lead to cleavage of the mRNA molecule and its degradation.
miRNA - microRNA - processed from stem-loop RNA precursors (pre-miRNAs) that are encoded within plant and animal genomes. The known functions of a few of these miRNAs indicate that they play widespread roles in growth and development. Animal miRNAs silence gene expression primarily by blocking the translation of mRNA transcripts into protein. They act as guides for a multiprotein complex, miRNA-induced silencing complex miRISC, which identifies mRNAs with imperfect complementarity in the 3' untranslated region of the message.
Dicer - dsRNA-specific endonuclease responsible for processing of the long targeting dsRNA (denoted the 'trigger') into siRNA or miRNA. This cleavage requires ATP and RDE-4 (a C. elegans Dicer homolog - RDE stands for RNAi defective), a protein containing two dsRNA binding domains. These intermediates of RNAi, siRNAs, are double stranded, and a 2 nt 3'-overhang is present in each sense and antisense strand of siRNA, due to the cleavage characteristics of Dicer. The 5' phosphate group of siRNA is maintained by a specific kinase; the free 3' hydroxyl group is essential for priming of the subsequent RdRP reaction (Nishikura, 2001).
Drosha and processing of microRNAs upstream of Dicer - Drosha converts primary miRNAs, most of which are full-length RNA polymerase II transcripts, into pre-miRNAs, 70 nt RNAs that fold into a stem-loop or hairpin structure. The RNase III family of double-stranded RNA-specific endonucleases is
characterized by the presence of a highly conserved 9 amino acid stretch in
their catalytic center known as the RNase III signature motif. The
drosha gene, encoding a new member of this family, was isolated
in Drosophila melanogaster.
Characterization of this gene revealed the presence of two RNase III signature
motifs in its sequence that may indicate that it is capable of forming an active
catalytic center as a monomer. The Drosha protein also contains an 825 amino
acid N-terminus with an unknown function. A search for the known homologues of
the Drosha protein revealed that it has a similarity to two adjacent annotated
genes identified during C. elegans genome sequencing. Analysis of the genomic
region of these genes by the Fgenesh program and sequencing of the EST cDNA
clone derived from it revealed that this region encodes only one gene. This
newly identified gene in nematode genome shares a high similarity to Drosophila
Drosha throughout its entire protein sequence. A potential Drosha homologue is
also found among the deposited human cDNA sequences. A comparison of these
Drosha proteins to other members of the RNase III family indicates that they
form a new group of proteins within this family (Filippov, 2000).
Hundreds of small RNAs of approximately 22 nucleotides, collectively named
microRNAs (miRNAs), have been discovered in animals and plants.
Although their functions are being unravelled, their mechanism of biogenesis
remains poorly understood. miRNAs are transcribed as long primary transcripts
(pri-miRNAs) whose maturation occurs through sequential processing events: the
nuclear processing of the pri-miRNAs into stem-loop precursors of approximately
70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into
mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate
nucleases, mediates the latter step, whereas the processing enzyme for the
former step is unknown. Another RNase III, human Drosha, has been identified as the
core nuclease that executes the initiation step of miRNA processing in the
nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro.
Furthermore, RNA interference of Drosha resulted in the strong accumulation of
pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two
RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing
of miRNAs, and have key roles in miRNA-mediated gene regulation in processes
such as development and differentiation (Lee, 2003).
MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory
genes that regulate the expression of homologous target-gene transcripts. They
have been implicated in the control of cell death and proliferation in flies,
haematopoietic lineage differentiation in mammals, neuronal patterning in
nematodes and leaf and flower development in plants. miRNAs are processed by the
RNA-mediated interference machinery. Drosha is an RNase III enzyme that has been
implicated in miRNA processing. Human Drosha is a
component of two multi-protein complexes. The larger complex contains multiple
classes of RNA-associated proteins including RNA helicases, proteins that bind
double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the
Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha
and the double-stranded-RNA-binding protein, DGCR8, the product of a gene
deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution
studies have revealed that both components of this smaller complex, termed
Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs
from the primary miRNA transcript (Gregory, 2004).
Mature microRNAs (miRNAs) are generated via a two-step processing
pathway to yield approximately 22-nucleotide small RNAs that regulate gene
expression at the post-transcriptional level. Initial cleavage is catalysed by
Drosha, a nuclease of the RNase III family, which acts on primary miRNA
transcripts (pri-miRNAs) in the nucleus. Here it is shown that Drosha exists in a
multiprotein complex, the Microprocessor, and begin the process of
deconstructing that complex into its constituent components. Along with Drosha,
the Microprocessor also contains Pasha (partner of Drosha), a double-stranded
RNA binding protein. Suppression of Pasha expression in Drosophila cells or
Caenorhabditis elegans interferes with pri-miRNA processing, leading to an
accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion
or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and
the appearance of phenotypic defects overlapping those observed upon examination
of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together,
these results indicate a role for Pasha in miRNA maturation and miRNA-mediated
gene regulation (Denli, 2004).
MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in
plants and animals. miRNAs are expressed in a
developmentally and tissue-specific manner and regulate the translational
efficiency and stability of partial or fully sequence-complementary mRNAs.
miRNAs are excised in a stepwise process from double-stranded RNA precursors
that are embedded in long RNA polymerase II primary transcripts (pri-miRNA).
Drosha RNase III catalyzes the first excision event, the release in the nucleus
of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to
the cytoplasm and further processing by Dicer to mature miRNAs.
The human DGCR8, the DiGeorge syndrome critical region gene 8, and
its Drosophila melanogaster homolog have been characterized. Biochemical and cell-based
readouts are provided to demonstrate the requirement of DGCR8 for the maturation of miRNA
primary transcripts. RNAi knockdown experiments of fly and human DGCR8 results
in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. These
results suggest that DGCR8 and Drosha interact in human cells and reside in a
functional pri-miRNA processing complex (Landthaler, 2004).
A critical step during human microRNA maturation is the processing of the
primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate
the 60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA
substrates and selects its cleavage sites has remained a mystery, especially
given that the known targets for Drosha processing show no discernable sequence
homology. Human Drosha selectively cleaves RNA hairpins bearing a large (10
nucleotides) terminal loop. From the junction of the loop and the adjacent stem,
Drosha then cleaves approximately two helical RNA turns into the stem to produce
the precursor microRNA. Beyond the precursor microRNA cleavage sites,
approximately one helix turn of stem extension is also essential for efficient
processing. While the sites of Drosha cleavage are determined largely by the
distance from the terminal loop, variations in stem structure and sequence
around the cleavage site can fine-tune the actual cleavage sites chosen (Zeng, 2005).
The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release approximately 70 nt pre-miRNAs that are subsequently processed by Dicer to generate mature approximately 22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. These study provides a novel example of eukaryotic gene organization and RNA-processing control (Kim, 2007).
MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a approximately 60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. From that duplex, the mature miRNA is incorporated into the silencing complex. This study identified an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. These pre-miRNAs/introns have been called 'mirtrons', and 14 mirtrons have been identified in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha (Ruby, 2007).
RdRP - RNA-directed RNA polymerase (SDE-1 in plants: EGO-1 in C. elegans) - provides amplification by several routes, such as replication of long trigger dsRNAs (similar to viral RNA replicases) or copying of short siRNAs (similar to the action of a known tomato RdRP) in a primer-independent manner - siRNA-primed RdRP reaction converts target mRNA into dsRNA, as well as possibly replicating trigger dsRNA (Nishikura, 2001).
How does an initial sub-stochiometric dose of dsRNA become converted into a sufficient quantity of siRNA to effectively carry out the process of RNAi? Studies show that repeated cycles of dsRNA synthesis and concomitant siRNA/primer production result in targeted mRNA degradation, and that this process can account for the underlying mechanism responsible for PTGS, quelling, and RNAi. Both single- and double-stranded RNAs can serve as templates for siRNA incorporation into dsRNA in Drosophila extracts. However, the rapid degradation of dsRNA suggests that amplification of just the trigger dsRNA is of limited value. RdRP-dependent as well as RDrP-independent mechanisms may be involved in the generation of dsRNA up to the full-length of the target RNA, according to one of the following schemes: (1) a single siRNA primer would be extended from various positions along individual template strands by RdRP to generate dsRNAs; and (2) different siRNAs would associate along a single template RNA and be extended by RdRP to the adjacent siRNA primer. The extension products would be ligated to
generate dsRNAs. This model would require RdRP activity as well as an RNA ligase step; and (3) dsRNAs would be formed by a primer 'guide' mechanism where they
would align along the template for subsequent ligation. All these mechanisms could generate dsRNA of sufficient length to be cleaved by RNase III-type activity since
this requires a minimum of 39 base pairs. The first and second models are favored since RdRP activity would be required to amplify the target dsRNA sufficiently when substiochiometric amounts of the trigger dsRNA are involved in initiating RNAi. The most convincing evidence for the involvement of RdRP activity in Drosophila RNAi comes from results using the synthetic 21 nucleotide GFP duplex siRNA where full-length GFP dsRNA was produced from a single primer. The extension of both strands of the synthetic siRNA in a template-dependent manner to yield the expected dsRNA products would specifically require an RdRP. The role for helicase activity in RNAi, as shown for qde-3 in Neurospora and SDE-3 in Arabidopsis in the genetic screens, may be to unwind the primers or the dsRNA trigger, but this remains to be demonstrated (Lipardi, 2001).
Other evidence, however, points to an absence of RdRP function in Drosophila. No member of this nearly ubiquitous family of polymerases has been detected by BLAST searching the nearly complete genome sequence of Drosophila or humans. Further, in Drosophila, the mRNA is cleaved only within the region of identity with the dsRNA (Zamore, 2000). In humans, the most compelling evidence against the involvement of an RdRP is the finding that siRNAs that connact act as primers for an RdRP because they contain blocked 3' termini nonetheless trigger efficient RNAi in vivo (Holen, 2002). Clearly, the potential role of RdRP in magnifying the RNAi reaction in Drosophila and mammals needs further investigation.
In Drosophila, two features of small interfering RNA (siRNA) structure -- 5' phosphates and 3' hydroxyls -- are reported to be essential for RNA interference (RNAi). As in Drosophila, a 5' phosphate is required for siRNA function in human HeLa cells. In contrast, no evidence was found in flies or humans for a role in RNAi for the siRNA 3' hydroxyl group. In vitro data suggest that in both flies and mammals, each siRNA guides endonucleolytic cleavage of the target RNA at a single site. It is concluded that the underlying mechanism of RNAi is conserved between flies and mammals and that RNA-dependent RNA polymerases are not required for RNAi in these organisms (Schwarz, 2002b).
Three models have been proposed for RNAi in Drosophila. Each model seeks to explain the mechanism by which siRNAs direct target RNA destruction. In one model, target destruction requires an RNA-dependent RNA polymerase (RdRP) to convert the target mRNA into dsRNA. The RdRP is hypothesized to use single-stranded siRNAs as primers for the target RNA-templated synthesis of complementary RNA (cRNA). The resulting cRNA/target RNA hybrid is proposed to then be cleaved by Dicer, destroying the mRNA and generating new siRNAs in the process. Key features of this model are that the ATP-dependent, dsRNA-specific endonuclease Dicer acts twice in the RNAi pathway, that target destruction should require nucleotide triphosphates to support the production of cRNA, and that a 3' hydroxyl group is essential for siRNA function, since siRNAs are proposed to serve as primers for new RNA synthesis. A second model proposes that single-stranded siRNAs do not act as primers for an RdRP, but instead assemble along the length of the target RNA and are then ligated together by an RNA ligase to generate cRNA. The cRNA/target RNA hybrid would then be destroyed by Dicer. This model predicts that target recognition and destruction should require ATP (or perhaps an NAD-derived high-energy cofactor) to catalyze ligation, as well as to support Dicer cleavage. Like the first model, the ligation hypothesis predicts that an siRNA 3' hydroxyl group should be required for RNAi. Furthermore, a 5' phosphate should be required for siRNA ligation, but ribonucleotide triphosphates other than ATP should not be required for target destruction. A third model hypothesizes that two distinct enzymes or enzyme complexes act in the RNAi pathway. As in the first model, Dicer is proposed to generate siRNAs from dsRNA. These siRNAs are then incorporated into a second enzyme complex, the RNA-induced silencing complex (RISC), in an ATP-dependent step or series of steps during which the siRNA duplex is unwound into single strands. The resulting single-stranded siRNA is proposed to guide the RISC to recognize and cleave the target RNA in a step or series of steps requiring no nucleotide cofactors whatsoever. The absence of a nucleotide triphosphate requirement for target recognition and cleavage is a key feature of this model (Schwarz, 2002b).
It has been demonstrated by two different experimental protocols that both recognition and endonucleolytic cleavage of a target RNA proceeds efficiently in the presence of less than 50 nM ATP, a concentration likely to be insufficient to support either the synthesis of new RNA or the ligation of multiple siRNAs into cRNA. However, the data also reveal an absolute requirement for a 5' phosphate for siRNAs to direct target RNA cleavage in Drosophila embryo lysates, a finding interpreted as reflecting an authentication step in the assembly of the RNAi-enzyme complex, the RISC. It was envisioned that the 5' phosphate is involved in obligatory noncovalent interactions with one or more protein components of the RNAi pathway. Nonetheless, the 5' phosphate requirement might formally reflect a requirement for the phosphate group in covalent interactions, such as the ligation of multiple siRNAs to generate cRNA (Schwarz, 2002b).
The mechanism of RNAi in flies and mammals has now been more fully defined by examining the requirement for a 5' phosphate and a 3' hydroxyl group on the antisense strand of the siRNA duplex. Initially, the role of these functional groups was examined in siRNA function in vitro, using both Drosophila and human cell-free systems that recapitulate siRNA-directed target RNA destruction. Then, these findings were validated in vivo in human HeLa cells. The data support a model for the RNAi pathway in which siRNAs function as guides for an endonuclease complex that mediates target RNA destruction. The requirement for a 5' phosphate is conserved between Drosophila and human cells and an siRNA 3' hydroxyl is dispensable in both systems. The data argue against an obligatory role for an RdRP in Drosophila or human RNAi, despite the clear requirement for such enzymes in PTGS in plants, quelling in Neurospora crassa, and RNAi in C. elegans and Dictyostelium discoideum. In this respect, the mechanism of RNAi in flies and mammals appears to be distinct from that of PTGS, quelling, and RNAi in worms and Dictyostelium, suggesting that the pathway in flies and mammals may be more restricted in the range of triggers that can elicit an RNAi response (Schwarz, 2002b).
The mechanism of RNA interference (RNAi) has been investigated in human cells. The status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells are phosphorylated, and 3' end biotin groups are not efficiently removed. No requirement was found for a perfect A-form helix in siRNA for interference effects, but an A-form structure is required for antisense-target RNA
duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen does not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results
suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells (Chiu, 2002).
RNA interference, a broadly used reverse genetics method in C. elegans, does not inhibit all genes. Loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system: neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens (Simmer, 2002).
Secondary siRNAs - The polarity of the RdRP reaction predicts that the newly synthesized dsRNA may extend beyond the sequence complementary to the initial trigger dsRNA, into upstream regions of the target mRNA (Nishikura, 2001).
Transitive RNAi - Secondary siRNAs were also able to induce secondary RNA interference, a phenomenon termed 'transitive RNAi" (Nishikura, 2001).
Initiation and maintenance - The initial step in gene silencing by RNAi, processing of the trigger dsRNA into siRNAs through the action of Dicer and other factors ('initiation'), is separable from the following amplification step involving RdRP ('maintenance'), but both steps are required for RNAi (Nishikura, 2001).
Enzymatic activity of Dicer - RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. The 218 kDa human Dicer has been cloned: its ribonuclease activity and dsRNA-binding properties have been characterized. The recombinant enzyme generates approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produces an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP is dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations is catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer binds dsRNA in vitro. Human Dicer expressed in mammalian cells colocalizes with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve understanding of RNA silencing and other Dicer-related processes (Provost, 2002).
Piwi/Argonaute/Zwille family consist of RNA endonucleases involved in mRNA cleavage - A gene that positively controls PTGS in Arabidopsis corresponds to a previously identified gene controlling development, AGO1. ago1 mutants display strong developmental alterations that affect plant architecture and fertility. The AGO1 protein shares similarity with a number of proteins containing Piwi and PAZ (Piwi/Argonaute/Zwille) domains: QDE-2, required for quelling in Neurospora; RDE-1, required for RNAi in C. elegans; eIF2C, presumed to play a role in the control of translation initiation in mammals; Aubergine/Sting is required for silencing of the repetitive Stellate locus in Drosophila and is required for the proper expression of the germline determinant Oskar; and Piwi, required for germline maintenance in Drosophila (Vaucheret, 2001).
The core protein component of all RISCs is a member of the Argonaute family of small RNA-guided RNA-binding proteins. The Drosophila genome encodes five Argonaute proteins, which form two subclades. The Ago subclade comprises Ago1 and Ago2, which bind miRNAs and siRNAs, respectively. Piwi, Aub, and Ago3 form the Piwi subclade of Argonaute proteins and bind repeat-associated siRNAs (rasiRNAs; also called piRNAs), which direct silencing of selfish genetic elements such as transposons.
Argonaute proteins are readily identified by their characteristic single-stranded RNA-binding PAZ domain and their Piwi domain, a structural homolog of the DNA-directed RNA endonuclease, RNase H (Song, 2004). The Piwi domain is thought to bind a small RNA guide both by coordinating its 5' phosphate and through contacts with the phosphate backbone, arraying the small RNA so as to create the seed sequence. Only a subset of Argonaute proteins contain Piwi domains that retain their RNA-directed RNA endonuclease activity: e.g., Ago1 in plants, Ago2 in mammals, and both Ago1 and Ago2 in flies. Drosophila Ago1 and Ago2 have been proposed to be restricted to the miRNA and siRNA pathways respectively. Such restriction of each class of small RNA to a distinct Argonaute complex could occur because miRNAs and siRNAs are produced by different Dicer pathways in flies (Forstemann, 2007).
miRNAs are cleaved from pre-miRNA by Dicer-1 (Dcr-1), acting with its dsRNA-binding protein partner, Loquacious (Loqs). siRNAs are produced from long dsRNA by Dicer-2 (Dcr-2), which partners with the dsRNA-binding protein R2D2. Thus, the different origins of miRNAs and siRNAs might direct them to distinct Argonaute proteins, with Dcr-1/Loqs recruiting Ago1 to miRNAs and Dcr-2/R2D2 directing siRNAs to Ago2. Alternatively, the specific structural differences between a miRNA/miRNA* duplex and an siRNA duplex might promote their sorting into Ago1- and Ago2-containing RISC, respectively. The Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2-RISC, promoting the incorporation of siRNAs and disfavoring the use of miRNAs as loading substrates for Drosophila Ago2. An independent mechanism acts in parallel to favor assembly of miRNA/miRNA* duplexes into Ago1-RISC and to exclude siRNAs from incorporation into Ago1. These two pathways compete for loading small-RNA duplexes with structures intermediate between that of an siRNA and a typical miRNA/miRNA* duplex, and such small RNAs partition between Ago1 and Ago2. Thus, small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures, rather than as a consequence of their distinct biogenesis pathways (Tomari, 2007).
RISC - RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (~22 nucleotides) derived from the double-stranded RNA trigger. RISC has been purified from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of ~500 kilodaltons. One constituent of this complex is a member of the Argonaute family of proteins. Dicer and AGO2 might physically interact, perhaps through their shared PAZ domains. Indeed, endogenous AGO2 can be coimmunoprecipitated with an epitope-tagged version of Dicer protein from transfected S2 cells, Dicer and RISC are biochemically separable, and none of the purified RISC fractions is able to process dsRNA into 22-nt fragments. One possibility is that Dicer is indeed a component of RISC but fails to process dsRNA when present in this complex. However, the current model is that the interaction between AGO2 and Dicer facilitates the incorporation of siRNAs into RISC complexes, which ultimately dissociate from Dicer and target cognate mRNAs for destruction (Hammond, 2001).
Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). A human biochemical system is described that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, it has been demonstrated that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting (Martinez, 2002).
It has been proposed that siRNAs act as primers for target RNA-templated dsRNA synthesis, even though homologs of the RNA-dependent RNA polymerases known to participate in gene silencing in other systems are apparently not encoded in D. melanogaster or mammalian genomes. Analysis of the fate of siRNA duplexes in the HeLa cell system does not provide evidence for such a siRNA-primed activity, but indicates that the predominant pathway for siRNA-mediated gene silencing is sequence-specific endonucleolytic target RNA degradation. Further evidence against siRNA-induced propagation of gene silencing in mammalian systems is that (1) the silenced gene returns to normal levels between 5 to 9 days posttransfection; (2) simultaneously expressed isoforms can be selectively targeted by siRNA duplexes (Martinez, 2002 and references therein).
In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5' cleavage products are readily detected for RNAi in vitro, only 3' cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5' and 3' cleavage products in a single experimental system. siRNA-programmed RISC is shown to generate both 5' and 3' cleavage products in vitro; cleavage requires Mg2+, but not Ca2+, and the cleavage product termini suggest a role for Mg2+ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn2+, but not by Ca2+ or Mg2+. It is proposed that during catalysis, a Mg2+ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified (Schwarz, 2004).
R2D2 and Argonaute-2, components of RISC in Drosophila - In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing, depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence, assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, that are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. These findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila (Okamura, 2004).
The RNAi pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). This process involves three proteins -- Dicer-2, R2D2, whose name derives from the fact that it contains two dsRNA-binding domains (R2) and is associated with DCR-2 (D2), and Argonaute-2, the core component of the RISC. The siRNA-generating enzyme was purified from Drosophila S2 cells and consists of two stoichiometric subunits: DCR-2 and the newly discovered R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer (carrying out the initiation step) to RISC, which carrys out the effector step (Liu, 2003). A model for RISC assembly is presented. First, R2D2 orients the Dcr-2/R2D2
heterodimer on the siRNA within the RISC-loading complex (RLC). As siRNA unwinding proceeds, the heterodimer is exchanged for Argonaute-2, the core component of the RISC. Indeed, single-stranded siRNA was not detected in the RLC assembled in
mutant ago2414 lysate. It is
hypothesized that unwinding occurs only when Ago2 is available, so that siRNA in the RLC is unwound only when the RISC can be assembled (Tomari, 2004).
Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. A Drosophila in vitro system was used in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro (Preall, 2006).
Fmr1 is a component of RISC in Drosophila: RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous
genes. This study reports the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs
(miRNAs). Two putative RNA-binding proteins, the Drosophila homolog of the mammalian fragile X mental retardation protein (FMRP), Fmr1, and VIG (Vasa intronic gene), are identified through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that Fmr1, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease (Caudy, 2002).
In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an
essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila.
Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway,
and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and
Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).
The influence of chromatin and methylation genes on PTGS: Maintenance silencing in plants - The Arabidopsis mutants ddm1 and met1 were isolated from a screen for mutations that result in a general reduction in methylation of the genome. MET1 encodes the major DNA methyltransferase. DDM1 encodes a protein related to SNF2/SWI2 chromatin-remodelling proteins. Both ddm1 and met1 mutants exhibit impaired PTGS, which correlates with a decrease in transgene methylation. The impairments of PTGS in ddm1 and met1 mutants differ: in ddm1 mutants PTGS is inhibited in the whole plant throughout its life, whereas in met1 mutants, PTGS is progressively inhibited during the course of plant development. This suggests that MET1 and DDM1 are involved in the maintenance and initiation steps of PTGS, respectively. Together, these results confirm the existence of a nuclear step in PTGS and reveal a genetic link between PTGS and TGS (Vaucheret, 2001). Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of polycomb dependent transcriptional gene silencing, indicating a connection between the two types of silencing (Pal-Bhadra, 2002).
In plants, RNA silencing is activated by viral RNAs that replicate via double-stranded intermediates and by transgenes with inverted repeat (IR) structures that produce dsRNA. Single-copy transgenes without IR structures also can activate RNA silencing. In these cases, it is possible that promoters in the transgene and the flanking plant DNA result in the transcription of both strands of the transgene DNA. However, it is likely as well that ssRNA is converted to dsRNA by an RNA-dependent RNA polymerase (RdRP); the activity of a putative RdRP encoded by the SDE1/SGS2 locus is required for RNA silencing in Arabidopsis. Virus-induced gene silencing (VIGS) is a type of RNA silencing that is initiated by virus vectors carrying portions of host genes. When plant transgenes are targeted by VIGS, sequence-specific methylation of the transgene DNA occurs. It is likely that this DNA methylation is directed by RNA-DNA interactions, because it is induced by RNA viruses that do not have DNA intermediates in their
replication cycles. The interacting RNA species in RNA-directed DNA methylation (RdDM) could be either dsRNA or siRNA derived from the initiator of silencing (Vaistij, 2002).
In some cases of VIGS, RNA silencing leads to the elimination of the viral RNA. However, despite the absence of the viral initiator, RNA silencing of the target gene persists. To explain this initiator-independent silencing, it has been proposed that there is a maintenance phase of RNA silencing that is distinct from initiation. Initiation requires the viral RNA initiator of silencing, whereas maintenance does not (Vaistij, 2002).
In Arabidopsis, these two phases are differentiated by mutation analysis. In wild-type plants, RNA silencing of a green fluorescent protein (GFP) transgene can be initiated by viruses and maintained in the absence of the viral initiator. However, in silencing-deficient (sde) mutants, RNA silencing can be initiated but the transition to maintenance does not occur. Initiation and maintenance also are observed as distinct processes when systemic RNA silencing is initiated by the localized delivery of ectopic DNA into Nicotiana benthamiana and tobacco. The systemic effect is caused by a signal that
moves through the plant, and the maintenance phase is inferred from the persistence of silencing after removal of the tissue that received the ectopic DNA. Maintenance of systemic silencing was associated with the methylation of the targeted gene (Vaistij, 2002).
It is likely that maintenance reflects the recruitment of the target gene or its RNA as
a source of dsRNA/siRNA; an initiator from the 5' or 3' part of a target GFP sequence causes systemic RNA silencing to be targeted along the entire length of the
transgene transcript. Correspondingly, the entire transcribed region of the GFP transgene is methylated. Thus, associated with the RNA silencing of a GFP transgene, there is a process that allows the influence of silencing to spread beyond the initiator sequence.
Target site spreading and maintenance both are features of GFP RNA silencing, but both of them are absent in the RNA silencing of two endogenous genes. Target site spreading is dependent on the SDE1/SGS2 putative RdRP and on the transcription of the target RNA. It is conclude that the maintenance of silencing involves the synthesis of dsRNA by SDE1/SGS2 using the full-length target RNA as a template (Vaistij, 2002).
In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for Arabidopsis mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily (Drosophila homolog: Okra). In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome (Kanno, 2004).
Whether DRD1 is involved in RNA-directed de novo methylation or acts to maintain RNA-induced non-CpG methylation remains to be determined. However, the heavy loss in drd1 plants of CpNpN methylation, which is not efficiently maintained in the absence of the RNA trigger, suggests a direct relationship between DRD1 activity and RNA signals. Given the relatedness of DRD1 to RAD54, it is intriguing to consider possible mechanistic similarities between RNA-directed DNA methylation and homologous DNA repair. In each case, the respective chromatin-remodeling factor could facilitate a homology search on duplex DNA, nucleosome displacement, and DNA unpairing and thereby allow heteroduplex formation and recruitment of enzyme complexes. In the RNA-directed DNA methylation pathway, this could create an RNA-DNA hybrid that attracts DMTases, thus accounting for the extraordinary specificity of cytosine methylation, which is largely restricted to the region of RNA-DNA sequence similarity. The lack of DRD1 homologs outside of the plant kingdom may mean that RNA-directed DNA methylation occurs only in plants. Alternatively, RAD54 or ATRX-like proteins may serve this function in other organisms (Kanno, 2004).
RNAi pathway and heterochromatin: In fission yeast, factors involved in the RNA interference (RNAi) pathway
including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for
heterochromatin assembly at centromeric repeats and the silent mating-type
region. RNA-induced initiation of transcriptional
gene silencing (RITS) complex containing the Argonaute protein and small
interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates
with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism
to couple siRNA generation with heterochromatin formation. The role were investigated of RNA-dependent
RNA polymerase (Rdp1) and its polymerase activity in
the assembly of heterochromatin. Rdp1, similar to RITS, localizes
to all known heterochromatic loci, and its localization at centromeric repeats
depends on components of RITS and Dicer as well as heterochromatin assembly
factors including Clr4/Suv39h and Swi6/HP1 proteins. A point
mutation within the catalytic domain of Rdp1 abolishes its RNA-dependent RNA
polymerase activity and results in the loss of transcriptional silencing and
heterochromatin at centromeres, together with defects in mitotic chromosome
segregation and telomere clustering. Moreover, the RITS complex in the rdp1
mutant does not contain siRNAs, and is delocalized from centromeres. These
results not only implicate Rdp1 as an essential component of a self-enforcing
RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase
activity in siRNA production necessary for heterochromatin formation (Sugiyama, 2005).
The establishment of centromeric heterochromatin in the fission yeast
Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway.
Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs)
that actively recruit components of heterochromatin to centromeres. Both
centromeric siRNAs and the heterochromatin component Chp1 are components of the
RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the
association of RITS with centromeres is linked to Dicer activity. In turn,
centromeric binding of RITS promotes Clr4-mediated methylation of histone H3
lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to
centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3
and is bound by Swi6. Chp1 associates with the mating type
locus and telomeres and Chp1 localization to heterochromatin depends on its
chromodomain and the C-terminal domain of the protein. Another protein component
of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with
Chp1 through the C-terminal domain of Chp1, and this interaction is necessary
for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein
component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and
Tas3 can still bind to noncentromeric loci, although their association with
centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex
that associates with noncentromeric heterochromatin independently of the RNAi
pathway (Petrie, 2005).
RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety
of eukaryotes to generate small interfering RNAs that target homologous
messenger RNA, resulting in their destruction. This process is widely used to
'knock-down' the expression of genes of interest to explore phenotypes. In
plants, fission yeast, ciliates, flies and mammalian cells, short interfering
RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous
genomic locus, which can result in transcriptional silencing or sequence
elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA
interactions at the homologous locus. Alternatively, they may act by
interactions between siRNA and nascent transcript. In fission
yeast (Schizosaccharomyces pombe), chromatin modifications are directed by
RNAi only if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA
polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA
pol II disrupts transcriptional silencing, indicating that, like other RNA
processing events, RNAi-directed chromatin modification is coupled to
transcription (Schramke, 2005).
stRNA - small temporally regulated RNAs - stRNAs of 22 nucleotides regulate timing of gene expression during development of the nematode C. elegans. This regulation occurs at a posttranscriptional, presumably translational. One of the two stRNAs, let-7, as well as its target gene, lin-41, are highly conserved even in humans, suggesting a wide employment of stRNA-mediated gene regulation. How downregulation is achieved through the putative RNA/RNA hybrid is, however, unclear. Evidence with lin-4 suggests that mRNA stability and polyadenylation level as well as translation initiation are not affected (Grosshans, 2002).
The Heterochronic pathway in C. elegans: lin-14, lin-28 and lin-41 are targets of stRNAs. Loss-of-function lin-14 mutations cause blast cells to skip the L1 stage and assume fates associated with L2, followed by normal development to the adult. In contrast, gain-of-function lin-14 alleles lead to a reiteration of L1 cell fates at later developmental stages. Similarly, lin-28 mutants indicate a role for this gene in L1/L2 stages to regulate L2/L3 cell fates. Loss-of-function lin-28 mutants have no effect during L1 but cause premature expression of L3 fates during L2, while gain-of-function lin-28 alleles cause cells to reiterate L2 fates at later larval stages. In lin-41 mutants, lateral hypodermal cells develop normally through L1 and L2, but prematurely adopt an adult fate at the L3/L4 molt. Overexpression of wild-type LIN-41 results in the opposite phenotype, leading to a reiteration of the larval hypodermal cell fate at the end of L4 rather than the normal switch to adult development. lin-14 and lin-28 mRNAs are translationally controlled the lin-4 heterochronic gene as a mediator of this regulation. lin-4 encodes two small RNAs, 61 and 22 nucleotides in length, with no apparent open reading frame. Gain-of-function lin-14 mutations map to the 3'-untranslated region (3'-UTR) of the lin-14 mRNA, a region with seven copies of a short sequence that could base pair with the lin-4 RNAs. lin-41 is a translational target of let-7.
Generality of the heterochronic pathway - There are 24 members of the RDE-1/AGO1/PIWI family in C. elegans. The degree of conservation between certain members of this family is striking. For example, ALG-1 and ALG-2 exhibit 41% identity with AGO1 from Arabidopsis and 67%-69% identity with AGO1 relatives in animals. The fact that divergent members of this family, including rde-1, qde-2, and ago-1, all function in gene silencing suggests that PTGS mechanisms represent an important ancestral function of genes within this family. One feature that emerges from studies of these developmental phenotypes OF RDE-1 genes is that many of these genes appear to regulate germ cell and stem cell functions. Perhaps germ cells and stem cells have developed PTGS mechanisms for suppressing viral and transposon pathogens that might otherwise degrade the genome and, thus, the totipotency of these cells. Alternatively, rde-1-related genes, alg-1 and alg-2, function with natural small RNA cofactors in specific developmental gene regulation events (Grishok, 2001).
Conservation of the heterochronic pathway in Drosophila - Expression of the Drosophila let-7 homolog rises at the end of the third larval instar in Drosophila in apparent synchrony with the ecdysone pulse, and maintained at high levels throughout pupal development. Several potential binding sites for the EcR/USP heterodimer map within 1-2 kb of the Drosophila let-7 gene, providing support for a direct regulatory
connection. The Drosophila genome also contains a gene that is similar to the let-7 target gene, lin-41 (CG1624/dappled/wech) with potential let-7 binding sites in its 3'-UTR. CG1624 mRNA is detected in NBs and GMCs of the ventral cord and brain and is expressed throughout NB lineage development (Brody, 2002). Homologs of LIN-28 and LIN-29 are also encoded by the fly genome. The discovery of these Drosophila genes, along with the potential ecdysone induction of fly let-7, provides exciting directions for future research. It will be interesting to determine if these genes have conserved their temporal identity functions from C. elegans to Drosophila (Thummel, 2002).
The silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila, has been analyzed. Both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) is sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline. It is concluded that homology-dependent gene silencing is used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated in controlling the expression of retrotransposons in the germline (Aravin, 2001).
Similarities between control of developmental timing by stRNAs and RNAi - lin-4 and let-7 RNAs both act as repressors of their respective target genes, lin-14, lin-28, and lin-41. Repression in all these cases requires the presence of stRNA complementary sequences in the 3' untranslated regions (UTRs) of the target mRNAs, suggesting that mRNA/stRNA hybrids can form. The presence of these stRNA-binding elements is indeed sufficient to confer stRNA-dependent expression on unrelated reporter genes. In addition to sharing a similar size and, presumably, mode of action, lin-4 and let-7 are also both synthesized as longer precursors that, in silico, can be folded into stem-loop structures. Processing into the mature forms involves the RNase III-like protein Dicer (dcr-1 in C. elegans) as well as the AGO1/rde-1 family members agl-1 and agl-2. This is another notable parallel to RNAi, where siRNA biogenesis likewise requires Dicer and distinct members of the AGO1/rde-1 family (Grosshans, 2002).
let-7 microRNA in a multiple-turnover RNAi enzyme complex - In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. In human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells (Hutvagner, 2002).
miRNP, a second protein complex containing an Argonaute homolog and micro RNAs - eIF2C (eukaryotic initiation factor 2C), is a member of the Argonaute proteins. A novel RNP has been identified that sediments as an ~15S particle on sucrose gradients and contains Gemin3, Gemin4, and human eIF2C2 along with numerous very small, single stranded cellular, ~22-nt RNAs (microRNAs, miRNAs). Gemin3 and Gemin4 may interact directly with eIF2C2 and have been identified as part of another multi-protein complex containing the Survival of Motor Neurons (SMN) protein, Gemin2, Gemin5, and Gemin6. The SMN-containing complex is distinct from the miRNP. The SMN complex functions in the assembly and restructuring of diverse RNP particles, including spliceosomal snRNPs. SMN protein is defective in the neurodegenerative disease, spinal muscular atrophy (SMA). It is, therefore, presumed that the underlying cause of SMA is a failure in snRNP biogenesis or recycling. Gemin3 is a 105-kD member of the DEAD-box family of putative ATP-dependent RNA helicases. Gemin3 may provide a catalytic function in the assembly of RNPs. The presence of eIF2C2 in the miRNP, establishes a first tentative link between the ~550-kD complex and the miRNA and RNAi pathways. miRNAs are tightly associated with the non-SMN, Gemin3-containing complex. In fact, this miRNP appears to be home to at least 40 different miRNAs, all but nine of them not identified in the original screen to clone and sequence human miRNAs. Like the original siRNAs, lin-4 and let-7, each of the new miRNAs is encoded in genomic sequence that can fold to form a ~70-nucleotide precursor RNA. It is suggested that miRNA precursors are processed in the cytoplasm by Dicer, which then passes the single-stranded miRNAs to the miRNP (Schwartz, 2002a).
Longer siRNAs contribute to systemic silencing and methylation of homologous DNA - RNA silencing is a eukaryotic genome defence system that involves processing of double-stranded RNA (dsRNA) into 21-26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA-directed methylation/transcriptional suppression of homologous genomic DNA. siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21-22 nt) and long (24-26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence-specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signaling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals (Hamilton, 2002).
In C. elegans, there is only partial overlap in the RNA silencing-related mechanisms that control retroelement activity and mediate dsRNA interference. Some mutations suppress both processes whereas others affect only one. Clearly there are degrees of complexity in RNA silencing that are not apparent from the well studied dsRNA/Dicer/RISC-based process. In plants there is probably a similar degree of complexity because both classes of siRNA are associated with transgene silencing, whereas retrotransposon siRNA belongs only to the long class. Similarly, of the four loci identified in a mutant screen as being required for transgene-induced RNA silencing, only sde4 has any effect on AtSN1 siRNA. Moreover, sgs2 and sde1, -3 and -4 interfered with transgene methylation, but only sde4 interfered with methylation of AtSN1 DNA. Presumably, the classes of siRNA or RNA silencing genes are not involved equally in different branches of the silencing pathway. Some of the complexity may simply be a result of compartmentalization with, for example, RNA turnover degradation in the cytoplasm and DNA methylation in the nucleus (Hamilton, 2002 and references therein).
Purification and characterization of RISC complex of Drosophila - RNA interference (RNAi) regulates gene expression by the cleavage of
messenger RNA, by mRNA degradation and by
preventing protein synthesis. These effects are mediated by a
ribonucleoprotein complex known as RISC 1(RNA-induced
silencing complex 1). Four Drosophila
components (short interfering RNAs, Argonaute 2, VIG and FXR3) of a RISC enzyme have been identified that degrade specific mRNAs
in response to a double-stranded-RNA trigger. Tudor-SN (tudor staphylococcal nuclease) -- a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain -- is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, purified Tudor-SN exhibits
nuclease activity similar to that of other staphylococcal nucleases.
Notably, both purified Tudor-SN and RISC are
inhibited by a specific competitive inhibitor of micrococcal
nuclease. Tudor-SN is the first RISC subunit to be identified
that contains a recognizable nuclease domain, and could therefore
contribute to the RNA degradation observed in RNAi (Caudy, 2003).
These data strongly indicate that Tudor-SN is a bona fide RISC
component. This is reflected by the co-purification of Tudor-SN and
RISC in Drosophila, C. elegans and mammalian cells. However, it
remains open to question whether Tudor-SN is a catalytic engine
of RNAi. Despite the aforementioned data, there are potential
inconsistencies. (1) Purified, recombinant Tudor-SN is
non-sequence-specific, in contrast to RISC, which shows a high degree
of selectivity for its mRNA targets. (2)Tudor-SN will
cleave both RNA and DNA, whereas no DNase activity is detected in RISC. (3) Several investigators have
detected specific cleavage of mRNAs within the siRNA-mRNA hybrid, and
this is difficult to rationalize with the known activities of
Tudor-SN and related enzymes. It is certainly consistent
with the biochemical data to suppose that RISC contains multiple
nucleases, only one of which (the putative Slicer) can catalyse
site-specific mRNA cleavage. In this scenario, Tudor-SN might act to
degrade the remainder of the mRNA. In accord with this idea,
targeting of an mRNA by a single siRNA often results in complete
degradation of the mRNA. Alternatively, it is possible that
Tudor-SN does not have a catalytic role in the RISC complex. Indeed,
pdTp is a competitive inhibitor that engages the potential
nucleic-acid-binding domains of Tudor-SN. Thus, inhibition of RISC by
pdTp may reflect a block in the ability of Tudor-SN to engage RNAs,
possibly including the mRNA target, in the context of the RISC
complex. Answers to these questions will come only from understanding
RISC in sufficient detail to allow reconstitution of its native
activity from purified components such that
the individual contributions of each to the varied roles of the RNAi
effector machinery can be studied in detail (Caudy, 2003).
Specificity of microRNA target selection in translational repression - MicroRNAs (miRNAs) are a class of noncoding RNAs found in organisms as evolutionarily distant as plants and mammals, yet most of the mRNAs they regulate are unknown. The ability of an miRNA to translationally repress a target mRNA is largely dictated by the free energy of binding of the first eight nucleotides in the 5' region of the miRNA. However, G:U wobble base-pairing in this region interferes with activity beyond that predicted on the basis of thermodynamic stability. Furthermore, an mRNA can be simultaneously repressed by more than one miRNA species. The level of repression achieved is dependent on both the amount of mRNA and the amount of available miRNA complexes. Thus, predicted miRNA:mRNA interactions must be viewed in the context of other potential interactions and cellular conditions (Doench, 2004).
Nine 3' UTRs were constructed, containing two binding sites each, that are predicted to base-pair to endogenous let-7a miRNA with varying DeltaG (free energy) values in the 5' region. Unlike results from previous experiments, these constructs do not contain flanking binding sites. let-7a was chosen because it is known to be highly expressed in HeLa cells, and paralogs expressed in HeLa cells share the same 8 nt in the 5' region. Again, the degree of repression correlated with the DeltaG values. However, under conditions of pairing with endogenous let-7a, construct D, with a free energy value of -6.3 kcal/mole, is essentially inactive for repression. This contrasts with previous results with transfected siRNAs in which values of -5 to -6 kcal/mole were active. To determine if this difference could be due to the concentration of miRNA, the experiment was repeated with additional let-7a introduced as an siRNA. As expected, additional let-7a did not lead to any repression of constructs with weak DeltaG values. Interestingly, only a modest increase in repression (38%) was observed for construct A, with the strongest DeltaG value (-11.0kcal/mole), yet for construct D, with a near-threshold DeltaG value of -6.3 kcal/mole, additional let-7a miRNA greatly increased repression (189%). Thus, miRNAs likely exist in a concentration-dependent association with their binding sites, and the presence of more miRNAs increases these interactions, resulting in more repression. This model predicts that increasing the amount of mRNA would have the opposite effect. Indeed, exchanging the weak herpes virus thymidine kinase promoter for the strong CMV promoter in the construct with four original CXCR4 siRNA sites led to a dramatic decrease in repression, from 12-fold to <4-fold (Doench, 2004).
The activity of the let-7a constructs also confirmed the detrimental effect of G:U wobble pairing. A construct with a strong DeltaG value, but with a G:U wobble at position 5, was not repressed with endogenous let-7a. Only upon addition of more let-7a could this construct be repressed. Furthermore, constructs with two G:U wobbles were not repressed by endogenous let-7a, nor did they significantly respond to additional let-7a (Doench, 2004).
The spacing requirements on the mRNA for miRNA interaction were examined. Constructs with four original CXCR4 siRNA sites were used, and the distance between the two internal sites was varied. 3' UTRs with the two internal CXCR4 sites spaced by 4 or 0 nt showed similar repression. To investigate possible steric hindrance between binding sites, constructs were designed such that the binding site for the 3' region of one CXCR4 siRNA would overlap with the binding site for the first four 5' nucleotides of another CXCR4 siRNA. To ensure that each internal site had a similar affinity for the miRNA, the binding site for the 3' region was disrupted in both sites. Perhaps surprisingly, this construct showed no decrease in repression. However, if this overlap between the two sites was increased to 9 nt, the construct gave the same amount of repression as only one internal site. Because a binding site can prevent access to a sufficiently close binding site, these results suggest that a factor stably associates with the mRNA. Indeed, miRNAs are thought to act by binding to their target mRNAs rather than by a catalytic mechanism requiring only a transient association between the miRNA and mRNA (Doench, 2004).
Combinatorial regulation, in which two factors simultaneously regulate a single gene, is a common feature of eukaryotic cells. To test if a single mRNA could be repressed by more than one miRNA, two 3'-UTR constructs were made, each of which contained two sites for the CXCR4 siRNA and two sites for a GFP siRNA. To avoid possible competition between the two siRNAs for access to protein assembly factors, the siRNAs were transfected at a less than saturating concentration (1 nM). The results indicate that two miRNAs can indeed simultaneously translationally repress a single mRNA. When either construct, GFP-CXCR4-CXCR4-GFP or CXCR4-GFP-GFP-CXCR4, was transfected with either siRNA alone, the degree of repression was approximately threefold. In contrast, cotransfection with both siRNAs results in approximately eightfold repression. Clearly, these reporters are being regulated by both siRNAs (Doench, 2004).
These studies on an endogenous miRNA, let-7a, indicate that a potential target must be evaluated in its cellular context. A binding site that is not repressed by endogenous levels of miRNA becomes repressed upon addition of exogenous miRNA. Thus, the level of expression of both the mRNA and the miRNA, as well as potential competing binding sites on other mRNAs, need to be taken into account to determine whether the mRNA is endogenously regulated by the miRNA. Validation of predicted miRNA:mRNA interactions by ectopic expression of either the mRNA target at artificially low levels, or the miRNA at artificially high levels, may 'confirm' an interaction that does not exist in vivo. It is well-established that many miRNAs are limited in their expression to certain stages in development or to certain tissues and cell types. Computational prediction would be aided by taking into consideration expression profiling of both miRNA and mRNA levels, and biochemical methods or genetic analysis may be needed for definitive proof of an miRNA:mRNA interaction (Doench, 2004).
This study brings into focus the question of miRNA specificity. Indeed, miRNAs are an abundant species of RNA both in terms of the sheer number of miRNAs in the genome, currently estimated at 200-255 for the human genome (Lai 2003), and in terms of their expression levels, as some miRNAs are expressed at >1000 copies per cell. Additional factors may also be important for determining in vivo targets of miRNAs, such as the FMRP protein, a known regulator of mRNA translation that has been implicated in RNA silencing complexes. Alternatively, specificity may be entirely dictated by the sequence of the miRNA itself. That the thermodynamic stability of a region spanning only 8 nt, a surprisingly low information content, is sufficient for miRNA activity may indicate a broad role for miRNAs in the regulation of gene expression (Doench, 2004).
K boxes and Bearded boxes, targets of post-transcriptional gene regulation - In Drosophila, two 3'-UTR sequence motifs, the K box (cUGUGAUa) and the Brd box (AGCUUUA) mediate negative post-transcriptional regulation. Although originally identified in the 3' UTRs of Notch pathway target genes encoding basic helix-loop-helix (bHLH) repressors and Bearded family proteins (see Bearded), modes of regulation mediated by both motifs are spatially and temporally ubiquitous. This suggests that at least some of the many other Drosophila transcripts that contain K boxes or Brd boxes in their 3' UTRs are also actively regulated by these motifs. Since RNA-binding proteins typically show relatively relaxed binding specificities, it was hypothesized that an RNA component might be involved in recognition of these highly constrained motifs. This was bolstered by the finding that another motif common to the 3' UTRs of many of the same Notch pathway target genes, the GY box (uGUCUUCC), is complementary to and mediates RNA duplex formation through the proneural box (AUGGAAGACAAU), a motif located in the 3' UTRs of transcripts encoding proneural bHLH activators, (Lai, 2002).
Drosophila miRNAs encoded by 11 of 21 distinct genomic miRNA loci are complementary to the K box at their 5' end, with all but miR-11 having a perfect (8/8) antisense match to the extended K box consensus (UAUCACAG). Notably, the most 5' nucleotide of miR-11 is a cytosine residue, making it complementary to the second most common nucleotide at this position in identified K boxes. In addition, perfect antisense matches to the Brd box and GY box were found at the 5' ends of fly miR-4 and fly miR-7, respectively. The precise complementarity of these miRNAs to K box, Brd box and GY box motifs suggests that they bind these sequences in 3' UTRs and, in the case of the former two motifs, mediate negative post-transcriptional regulation. Complementarity between miRNAs and 3' UTRs extends beyond core sequence motifs in many cases, providing additional support for the existence of the proposed RNA duplexes. Examples exist of extended miRNA complementarity to 3' UTRs containing K boxes, Brd boxes and GY boxes. Complements to all three sequence motifs are located exclusively at the 5' ends of miRNA, suggesting that some aspect of regulation may be shared by these different miRNAs. For example, a common factor might be involved in the recognition or stabilization of these short miRNA-3' UTR duplexes (Lai, 2002).
Several miRNAs complementary to K boxes (miR-11 and the miR-2b and miR-13 subfamilies) are broadly expressed throughout Drosophila development, consistent with their proposed involvement in temporally ubiquitous regulation mediated by K boxes; the GY box-complementary miRNA miR-7 is similarly broadly expressed during development. The expression of the single identified Brd box-complementary miRNA miR-4 is restricted to embryogenesis. However, since the search for miRNAs has not yet been saturating, other miRNAs complementary to Brd boxes that are expressed later in development might yet be found (Lai, 2002).
The regulatory role of the K box and Brd box in other organisms has not yet been tested. Nevertheless, the presence of their complements in worm and human miRNAs suggests that these modes of regulation have potentially been conserved. Notably, the complements to these motifs are also located specifically at the 5' ends of miRNA. The restricted location of complements in these different species further suggests that the regulatory targets of many other miRNAs will be determined by the sequence of their 5' ends. In agreement with this idea, most of the known lin-4 and let-7 target sequences also involve perfect complements with the 5' ends of these miRNAs. Systematic searches for the complements of other 5' miRNA ends in 3' UTRs may therefore identify new post-transcriptional regulatory sequence elements. It should be noted, however, that despite the existence of three conserved sites in the lin-14 3' UTR that include perfect complements to lin-4, normal regulation of lin-14 actually depends on variant lin-4 binding sites containing a bulged nucleotide in the 5' complementary region. Thus, this rule is probably not absolute (Lai, 2002).
Initially, miRNAs are transcribed as RNAs of approximately 70 nt containing a stem-loop structure; these are cleaved by the RNAse III enzyme Dicer to generate the mature miRNA. Curiously, only a single strand of the duplex precursor stem structure is generally stable and is recovered as miRNA. The model proposed here may help to explain this phenomenon, since the strand that is complementary to these identified 3' UTR motifs is nearly exclusively the one that is isolated as miRNA. The single exception is miR-5, whose sequence contains a K box. Notably, miR-5 and the K box-complementary miRNAs miR-6-1,2,3 (whose loci are incidentally located next to each other in the genome) are complementary at 20 of 21 continuous nucleotide positions. This suggests that miR-5 might influence or possibly interfere with the ability of miR-6-1,2,3 to interact with 3' UTRs that contain K boxes (Lai, 2002).
Negative regulation by K box- and Brd box-complementary miRNA must differ from lin-4-mediated regulation, because K boxes and Brd boxes have significant, though distinct, effects on both transcript stability and translational efficiency, whereas lin-4 is thought to act at a step following translational initiation. The GY box does not seem to have a strong effect at the cis-regulatory level. Other miRNAs may show additional regulatory capacities; efforts are underway to understand the different molecular mechanisms of regulation mediated by miRNA-3' UTR RNA duplexes (Lai, 2002).
Microbodies - subcellular sites of mRNA degradation - Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis remain obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. This study demonstrates that Argonaute proteins -- the signature components of the RNA interference (RNAi) effector complex, RISC -- localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis (Liu, 2005).
RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centers that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centers is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function (Sen, 2005).
A crucial role for decapping complex in miRNA-mediated gene silencing - In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex [consisting of DCP1 (CG11183) and DCP2 (CG6169)] and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins that are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions on the expression of reporter constructs were analyzed, allowing the monitoring specifically of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).
The precise molecular mechanism by which these proteins participate in the miRNA pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, these results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).
The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila Regulation of miRNA expression during neural cell specification -
MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. The regulation was studied of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. Significant differences are observed in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes marked lineage specificity was found for each of the miRNA in this study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir-124, mir-128). In contrast, mir-23, a miRNA previously implicated in neural specification, is restricted to astrocytes. mir-26 and mir-29 are more strongly expressed in astrocytes than neurons -- others are more evenly distributed (mir-9, mir-125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir-125 and mir-128). miRNA-mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA are strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development (Smirnova, 2005).
Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth -
Although hundreds of evolutionarily conserved microRNAs have been discovered, the functions of most remain unknown. This study describes the embryonic spatiotemporal expression profile, transcriptional regulation, and loss-of-function phenotype of Drosophila miR-1 (DmiR-1). DmiR-1 RNA is highly expressed throughout the mesoderm of early embryos and subsequently in somatic, visceral, and pharyngeal muscles, and the dorsal vessel. The expression of DmiR-1 is controlled by the Twist and Mef2 transcription factors. DmiR-1KO mutants, generated using ends-in gene targeting, die as small, immobilized second instar larvae with severely deformed musculature. This lethality is rescued when a DmiR-1 transgene is expressed specifically in the mesoderm and muscle. Strikingly, feeding triggers DmiR-1KO-associated paralysis and death; starved first instar DmiR-1KO larvae are essentially normal. Thus, DmiR-1 is not required for the formation or physiological function of the larval musculature, but is required for the dramatic post-mitotic growth of larval muscle (Sokol, 2005).
Aravin, A. A., Naumova, N. M., Tulin, A. V., Vagin, V. V., Rozovsky, Y. M. and Gvozdev, V. A. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr. Biol. 11(13): 1017-27. 11470406
Brody, T., Stivers, C., Nagle, J., Odenwald, W. F. (2002). Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen. Mech. Dev. 113(1): 41-59. 11900973
Caudy, A. A., Myers, M., Hannon, G. J. and Hammond, S. M. (2002). Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16: 2491-2496. 12368260
Caudy, A. A., et al. (2003). A micrococcal nuclease homologue in RNAi effector complexes. Nature 425(6956): 411-414. 14508492
Chiu, Y.-L. and Rana, T. M. (2002). RNAi in human cells: Basic structural and functional features of small interfering RNA. Molec. Cell 10: 549-561. 12408823
Czech, B. and Hannon, G. J. (2011). Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 12: 19-31. PubMed ID: 21116305
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. and Hannon, G. J. (2004). Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014): 231-5. 15531879
Doench, J. G. and Sharp, P. A. (2004). Specificity of microRNA target selection in translational repression. Genes Dev. 18: 504-511. 15014042
Filippov, V., Solovyev, V., Filippova, M., Gill , S. S. (2000). A
novel type of RNase III family proteins in eukaryotes. Gene 245(1): 213-21.
10713462
Forstemann, K., Horwich, M. D., Wee, L., Tomari, Y. and Zamore, P. D. (2007). Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1.
Cell 130(2): 287-97. Medline abstract: 17662943
Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T. and Doratotaj, B.
(2004) The Microprocessor complex mediates the genesis of microRNAs. Nature 432:
235240. 15531877
Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G. and Mello, C. C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106(1): 23-34. 11461699
Grosshans, H. and Slack, F. J. (2002). Micro-RNAs : small is plentiful. J. of Cell Biology 156: 17-22. 11781331
Hamilton, A., et al. (2002). Two classes of short interfering RNA in RNA silencing. EMBO J. 21: 4671-4679. 12198169
Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. and Hannon, G. J. (2001).
Argonaute2, a link between genetic and biochemical analyses of RNAi.
Science 93(5532): 1146-50. 11498593
Holen, T. et al. (2002). Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30(8): 1757-66. 11937629
Hutvagner, G. and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297: 2056-2060. 12154197
Ishizuka, A., Siomi, M. C. and Siomi1, H. (2002). A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16: 2497-2508. 12368261
Kanno, T., et al. (2004). Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation.
Curr. Biol. 14: 801-805. 15120073
Kim, Y. K. and Kim, V. N. (2007). Processing of intronic microRNAs.
EMBO J. 26(3): 775-83. Medline abstract: 17255951
Lai, E. C. (2002). Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30(4): 363-4. 11896390
Landthaler, M., Yalcin, A. and Tuschl, T. (2004). The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for
miRNA biogenesis. Curr. Biol. 14(23): 2162-7. 1558916
Lee, Y., Ahn, C., Han, J., Choi, H. and Kim, J. (2003). The nuclear RNase III Drosha
initiates microRNA processing. Nature 425: 415419. 14508493
Lipardi, C., Wei, Q. and Paterson. B. M. (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107: 297-307. 11701121
Liu, J., Valencia-Sanchez, M. A., Hannon, G. J., and Parker, R. (2005).
MicroRNA-dependent localization of targeted mRNAs to mammalian
P-bodies. Nat. Cell Biol. 7: 719-723. 15937477
Martinez, J., et al. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110: 563-574. 12230974
Mourelatos, Z., Dostie, J., Paushkin, S., Sharma, A., Charroux, B., Abel, L., Rappsilber, J., Mann, M. and Dreyfuss, G (2002). miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16(6): 720-8. 11914277
Nishikura, K. (2001). A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 16: 107(4): 415-8. 11719182
Okamura. K., Ishizuka, A., Siomi, H. and Siomi, M. C. (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18(14): 1655-66. 15231716
Okamura, K., Hagen, J. W., Duan, H., Tyler, D. M. and Lai, E. C. (2007).
The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila.
Cell 130(1): 89-100. Medline abstract: 17599402
Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (2002). RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol. Cell 9(2): 315-27. 11864605
Petrie, V. J., et al. (2005). RNA interference (RNAi)-dependent and
RNAi-independent association of the Chp1 chromodomain protein with distinct
heterochromatic loci in fission yeast. Mol. Cell. Biol. 25(6): 2331-46.
15743828
Preall, J. B., He, Z., Gorra, J. M. and Sontheimer, E. J. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol. 16(5): 530-5. 16527750
Provost, P., Dishart, D., Doucet, J., Frendewey, D., Samuelsson, B., and Radmark, O. (2002). Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 21: 5864-5874. 12411504
Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (2005). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11(11): 1640-7. 16177138
Ruby, J. G., Jan, C. H. and Bartel, D. P. (2007). Intronic microRNA precursors that bypass Drosha processing. Nature 448(7149): 83-6. Medline abstract: 17589500
Schramke, V., et al. (2005). RNA-interference-directed chromatin modification
coupled to RNA polymerase II transcription. Nature 435(7046): 1275-9. 15965464
Schwarz, D. S. and Zamore, P. D. (2002). Why do miRNAs live in the miRNP?
Genes Dev. 16: 1025-1031. 12000786
Schwarz, D. S., et al. (2002b). Evidence that siRNAs function as guides, not primers, in the Drosophila and Human RNAi pathways. Molec. Cell 10: 537-548. 12408822
Schwarz, D. S., Tomari, Y. and Zamore, P. D. (2004). The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr. Biol. 14: 787-791. 15120070
Sempere, L. F., Dubrovsky, E. B., Dubrovskaya, V..A, Berger, E. M., and Ambros, V. (2002). The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster. Dev. Biol. 244(1): 170-9. 11900466
Sen, G. L. and Blau, H. M. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell
Biol. 7: 633-636. 15908945
Simmer, F., et al. (2002). Loss of the putative RNA-directed RNA polymerase RRF-3 Makes C. elegans hypersensitive to RNAi. Curr. Biol. 12: 1317-1319. 12176360
Smirnova, L., Grafe, A., Seiler, A., Schumacher, S., Nitsch, R., Wulczyn, F. G. (2005). Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21(6): 1469-77. 15845075
Sokol, N. S. and Ambros, V. (2005). Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth. Genes Dev. 19: 2343-2354. 16166373
Song, J. J., Smith, S. K., Hannon, G. J. and Joshua-Tor, L. (2004). Crystal structure of Argonaute and its implications for RISC slicer activity, Science 305: 1434-1437. Medline abstract: 15284453
Sugiyama, T., Cam, H., Verdel, A., Moazed, D. and Grewal, S. I. (2005). RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling
heterochromatin assembly to siRNA production. Proc. Natl. Acad. Sci. 102(1):152-7. 1561584
Thummel, C. S. (2001). Molecular Mechanisms of Developmental Timing in C. elegans and Drosophila. Developmental Cell 1: 453-465. 11703937
Tomari, Y., Matranga, C., Haley, B., Martinez, N. and Zamore, P. D. (2004). A protein sensor for siRNA asymmetry. Science 306(5700): 1377-80. 15550672
Tomari, Y., Du, T. and Zamore, P.D. (2007). Sorting of Drosophila small silencing RNAs. Cell 130(2): 299-308. Medline abstract: 17662944
Vaistij, F. E., Jones, L. and Baulcombe, D. C. (2002). Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase Plant Cell 14: 857-867. 11971140
Vaucheret H, Beclin C, Fagard M. (2001). Post-transcriptional gene silencing in plants. J Cell Sci. 114: 3083-91 - Full text link
Zamore, P.D., et al. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1): 25-33. 10778853
Zeng, Y., Yi, R. and Cullen, B. R. (2005), Recognition and cleavage of
primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J.
24(1): 138-48. 15565168
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.