The Interactive Fly
Zygotically transcribed genes
INDEX
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).
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).
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).
RNA-directed RNA polymerase - 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).
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).
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).
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).
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).
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).
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