InteractiveFly: GeneBrief

bicoid stability factor: Biological Overview | References

BIOLOGICAL OVERVIEW

ClpXP is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX. Although it has been proposed that ClpXP is required for the mitochondrial unfolded protein response, additional roles for ClpXP in mitochondrial biogenesis are unclear. This study found that Drosophila leucine-rich pentatricopeptide repeat domain-containing protein 1 (DmLRPPRC1; Bicoid stability factor) is a specific substrate of ClpXP. Depletion or introduction of catalytically inactive mutation of ClpP increases DmLRPPRC1 and causes non-uniform increases of mitochondrial mRNAs, accumulation of some unprocessed mitochondrial transcripts, and modest repression of mitochondrial translation in Drosophila Schneider S2 cells. Moreover, DmLRPPRC1 over-expression induces the phenotypes similar to those observed when ClpP is depleted. Taken together, ClpXP regulates mitochondrial gene expression by changing the protein level of DmLRPPRC1 in Drosophila Schneider S2 cells (Matsushima, 2017).

ClpXP, Lon and m-AAA are ATP-dependent proteases that contribute to the degradation of all mitochondrial matrix proteins. ClpXP and Lon localize to the matrix space whereas the m-AAA protease is anchored in the membrane with its catalytic site exposed to the matrix space. ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain (Matsushima, 2017).

ClpXP is a barrel-shaped, hetero-oligomeric complex in which ClpP forms a two-stack heptameric ring-shaped structure to which two hexameric ClpX rings bind on each side. Lon also carries an AAA+ domain and forms homo-oligomeric, ring-shaped complexes. Lon and ClpP contain serine protease domains and these proteases are well conserved among species. Interestingly, mutations in each protease cause different diseases. Mutations in the gene encoding ClpP cause sensorineural hearing loss and ovarian failure in Perrault syndrome, while mutations in the genes encoding mitochondrial Lon cause cerebral, ocular, dental, auricular, skeletal anomalies (CODAS) syndrome. Moreover, a ClpP knock out in the mouse results in a phenotype similar to the relatively modest phenotype observed in patients with Perrault syndrome whereas deletion of the mitochondrial Lon protease gene causes early lethality (Matsushima, 2017).

ClpXP and Lon function mainly to degrade misfolded or damaged proteins (for example, proteins damaged by oxidation) to prevent the cell from accumulating defective proteins. These protein degradation pathways are collectively called protein quality control. Although there is some overlap in the substrate specificities of mitochondrial proteases, it has been proposed that some mitochondrial matrix proteins are specific substrates for each of them. However, to date, a limited number of proteins has been shown to be specific substrates for each. In addition, ClpXP and Lon also play a key role in mitochondrial biological processes. For instance, it has been shown previously that Lon regulates mitochondrial DNA (mtDNA) transcription by regulating the ratio of mitochondrial transcription factor A (TFAM) to mtDNA via degradation of TFAM. In addition, ClpXP modulates mitochondrial unfolded protein responses in C. elegans and mammals (Matsushima, 2017).

Metazoan mtDNA is transcribed as precursor polycistronic RNAs containing rRNAs, tRNAs, and mRNAs. After RNA processing and maturation, each RNA contributes to mitochondrial translation. The pentatricopeptide repeat (PPR) domain is a RNA binding domain and, in metazoans, the PPR protein family participates in mitochondrial RNA biogenesis. Leucine-rich pentatricopeptide repeat domain–containing proteins, called LRPPRCs, have multiple PPR domains. Both mammalian LRPPRC and its Drosophila melanogaster homologue, DmLRPPRC1 are required for polyadenylation of mitochondrial mRNAs (mt-mRNAs). LRPPRCs also control mt-mRNA stability, as loss of LRPPRC or DmLRPPRC1 results in a non-uniform reduction of mitochondrial mRNA abundance. LRPPRC interacts with the non-PPR RNA binding protein, SRA stem-loop interacting RNA binding protein (SLIRP) in ribonucleoprotein complexes. Interestingly, LRPPRC and SLIRP are degraded in mitochondria under some experimental conditions such as inhibition of mitochondrial transcription (Matsushima, 2017).

This study investigated the role of ClpXP protease in regulating the abundance of mitochondrial mRNA, and processing and translation of mitochondrial RNA in Drosophila cells. The results argue strongly that ClpXP modulates mitochondrial RNA biogenesis by the selective degradation of DmLRPPRC1 (Matsushima, 2017).

Mitochondrial transcripts were increased in ClpP knockdown cells, which may result from upregulation of mitochondrial transcription activity because mitochondrial tRNAs, which can be indicators of mitochondrial transcription activity, were increased in addition to upregulation of mtTFB2 protein and mRNA. Previously work that mitochondrial transcripts are also increased in Lon knockdown cells in association with an increase in mtTFB2 protein. These similar events might suggest that upregulation of mtTFB2 is a common response to defects in mitochondrial matrix protein turnover caused by depletion of either Lon or ClpXP. Moreover, mtDNA copy number is also increased in ClpP knockdown cells. These results might suggest that the increase in mtDNA copy number results from the increased levels of mitochondrial transcripts that are required for the synthesis of RNA primers during replication of mtDNA, as was reported previously. Another possible explanation is that the increase in mtDNA is caused by accumulation of other mtDNA replication factors by either direct or indirect effects of ClpP knockdown (Matsushima, 2017).

DmLRPPRC1 overexpression did not change the abundance of mitochondrial tRNAs. Therefore, DmLRPPRC1 may not be involved in mitochondrial transcription, which is in agreement with previous studies showing no link between LRPPRC and transcriptional activity. It has been shown that depletion of DmLRPPRC1 results in the reduction of mitochondrial mRNA levelsand the same phenomenon was observed also in vertebrate cells. In addition, inhibition of mitochondrial transcription reduces LRPPRC and SLIRP levels in vertebrate cells. These results indicate that levels of LRPPRC, SLIRP and mt-mRNAs are interdependent. Likewise, the current study showed that DmLRPPRC1 degradation is facilitated in some conditions, such as inhibition of mitochondrial transcription or depletion of DmSLIRP1 in Drosophila cells. Conversely, DmLRPPRC1 increased when ClpP or ClpX was knocked down, suggesting that DmLRPPRC1 is degraded by ClpXP. In agreement with this hypothesis, ClpP depletion prevented the facilitated degradation of DmLRPPRC1 caused by mitochondrial transcription inhibition or DmSLIRP1 knockdown. DmLRPPRC1 may be specifically degraded by ClpXP because Lon-depletion did not accumulate DmLRPPRC1 or inhibit the facilitated degradation of DmLRPPRC1. Moreover, this study showed that ClpX, which recognize the protein substrates of ClpXP, physically interacts with DmLRPPRC1 in the cells. Collectively, these results strongly suggest that DmLRPPRC1 is a specific substrate of ClpXP in Drosophila cells. By contrast, ClpP knockdown did not prevent the degradation of DmSLIRP1 caused by mitochondrial transcription inhibition or DmLRPPRC1 knockdown, which suggests that the facilitated degradation of DmSLIRP1 is not specifically mediated by ClpXP. DmLRPPRC1 and DmSLIRP1 stabilize each other. It is considered that ClpP knockdown increased DmSLIRP1 protein in control cells due to the DmLRPPRC1 accumulation (Matsushima, 2017).

The preferred amino acid sequences recognized by mitochondrial ClpXP remain unclear. At the same time, proteomic studies of E. coli ClpXP identified some classes of peptide sequences, called preferred degradation tags, and most of the preferred C-terminal degradation tags contain alanine in the C-terminal position. Interestingly, the amino acid in the C-terminal position of DmLRPPRC1 is alanine, suggesting that mitochondrial ClpXP might also recognize the alanine. These results show the possibility that in addition to LONP1, ClpXP may also contribute to the degradation of LRPPRC protein. However, unlike the situation in Schneider S2 cells, ClpXP is not the dominant protease in the degradation of LRPPRC protein in HeLa cells. Moreover, a recent report indicated that knockdown of Lon protease inhibits LRPPRC degradation in mouse cells. Because the C-terminal amino acid in mouse and human LRPPRC is not alanine, these differences in the substrate preference of proteases between species might arise from differential recognition of C-terminal degradation tags (Matsushima, 2017).

It has been shown that overexpressing ClpX in mouse C2C12 cells results in an increase in LRPPRC protein possibly involving the mitochondrial unfolded protein response pathway. This indicates that changes in the protein levels of ClpX may affect the gene expression of LRPPRC in mammalian cells. This study has shown that knockdown of ClpX or ClpP results in an increase of DmLRPPRC1 protein without an increase in the corresponding mRNA. These data indicate that in ClpP or ClpX knockdown Schneider S2 cells, the increase of DmLRPPRC1 likely does not result from signal transduction events (Matsushima, 2017).

This study showed that two unprocessed mRNAs, COXIII-ATP6/8 and ND6-CytB, are accumulated in ClpP knockdown cells or DmLRPPRC1 overexpression cells. Metazoan mtDNA is transcribed as precursor polycistronic transcripts containing mt-mRNAs, mt-rRNAs and mt-tRNAs. Most of the mt-mRNAs and mt-rRNAs are produced when mt-tRNAs are excised by ELAC2 and the RNaseP-complex. However, some processing sites are cleaved without an accompanying mt-tRNA excision, such as COXIII-ATP6/8 and ND6-CytB in Drosophila. The mechanisms involved in these cleavages remain largely unknown. In human cells, the ELAC2 and RNaseP-complexes do not appear to be involved in non-tRNA processing. Recent studies suggest that pentatricopeptide repeat domain 2 protein, which has one PPR domain, contributes to non-tRNA cleavages. DmLRPPRC1 contains multiple PPR domains. Excess DmLRPPRC1 protein may compete with canonical proteins performing the non-tRNA processing of COXIII-ATP6/8 and ND6-CytB (Matsushima, 2017).

An increase of DmLRPPRC1 protein results in a non-uniform increase in mitochondrial mRNA expression. Similar to the previously reported functions of vertebrate LRPPRC, DmLRPPRC1 is responsible for polyadenylation of mitochondrial mRNAs, which affects the stability of mt-mRNAs. However, this study did not detect any difference in migration of mt-mRNAs in northern blot analysis in ClpP RNAi cells. These results suggest that the length of poly(A) tails of mt-mRNAs are not changed dramatically in the presence of excess DmLRPPRC1 protein (Matsushima, 2017).

Excess DmLRPPRC1 resulted in a reduction of mitochondrial translation. These results are consistent a previous finding that shown that depletion of DmLRPPRC1 causes an increase in mitochondrial translation in flies. Similar to the current results, in Schizosaccharomyces pombe, overexpression of a multiple PPR domain-containing protein, PPR5, resulted in inhibition of mitochondrial protein synthesis without an obvious change in steady state levels of mitochondrial transcripts. Mitochondrial protein synthesis is reduced more severely in DmLRPPRC1 overexpressing cells although levels of mitochondrial mRNAs do not differ between ClpP knockdown cells and DmLRPPRC1 overexpression cells. DmLRPPRC1 was increased about two-fold in the former cells while about eight-fold in the latter. A previous study showed that the ratio of TFAM to mtDNA is important for the regulation of mitochondrial transcription and a higher ratio of TFAM to mtDNA causes a reduction in mitochondrial transcript abundance. Analogously, the ratio of DmLRPPRC1 to mtRNAs may be critical for the regulation of mitochondrial translation (Matsushima, 2017).

Ribosome profiling results showed that DmLRPPRC1 overexpression inhibited the formation of fully assembled 55S ribosomes, and enhanced the accumulation of mt-mRNAs in the lower-density fractions that contain mitochondrial small ribosomal subunits. In mitochondrial translation, mt-mRNAs initially interact with the small ribosomal subunit and then are assembled into the full ribosome. Therefore, the results suggest that excess DmLRPPRC1 does not prevent this interaction, but instead interferes with the assembly of mt-mRNA-small ribosomal subunit complexes with large ribosomal subunits (Matsushima, 2017).

In humans, recessive mutations of ClpP have been known to cause Perrault syndrome characterized by sensorineural hearing loss and ovarian failure. Similar to humans, ClpP knockout mice show hearing loss and infertility. Interestingly, knockout of ClpP also causes ineffective mitochondrial translation. A very recent study shows that ClpXP regulates the protein levels of ERAL1, a putative 12S rRNA chaperone. ERAL1 protein was accumulated in ClpP deficient mouse heart and the excess ERAL1 cause ineffective mitochondrial translation by the inhibition of mitochondrial ribosome assembly. To date, many proteins are on the list of candidates of ClpXP substrates. Therefore, some substrate proteins of ClpXP may be involved in mitochondrial translation. Further studies are necessary to clarify the relationship between protein substrates of ClpXP and mitochondrial translation (Matsushima, 2017).

In summary, it is concluded that DmLRPPRC1 is a specific substrate of ClpXP. An increase in DmLRPPRC1 protein, which can result from ClpXP depletion, causes non-uniform increases of mitochondrial mRNAs, accumulation of some unprocessed mitochondrial transcripts and partial inhibition of mitochondrial translation (Matsushima, 2017).

RNA sequences required for the noncoding function of oskar RNA also mediate regulation of Oskar protein expression by Bicoid Stability Factor

The Drosophila oskar (osk) mRNA is unusual in having both coding and noncoding functions. As an mRNA, osk encodes a protein which is deployed specifically at the posterior of the oocyte. This spatially-restricted deployment relies on a program of mRNA localization and both repression and activation of translation, all dependent on regulatory elements located primarily in the 3' untranslated region (UTR) of the mRNA. The 3' UTR also mediates the noncoding function of osk, which is essential for progression through oogenesis. Mutations which most strongly disrupt the noncoding function are positioned in a short region (the C region) near the 3' end of the mRNA, in close proximity to elements required for activation of translation. Bicoid Stability Factor (BSF) binds specifically to the C region of the mRNA. Both knockdown of bsf and mutation of BSF binding sites in osk mRNA have the same consequences: Osk expression is largely eliminated late in oogenesis, with both mRNA localization and translation disrupted. Although the C region of the osk 3′ UTR is required for the noncoding function, BSF binding does not appear to be essential for that function (Ryu, 2015).

One way to categorize RNAs is by their coding potential, or lack thereof. Members of one group, the mRNAs, have long open reading frames and are translated, thereby performing a coding function. The other group, consisting of RNAs without long open reading frames, has many members with no consistent size or organization. Such noncoding RNAs perform a wide variety of structural, regulatory and enzymatic functions. Often, these coding and noncoding roles are mutually exclusive. Most of the exceptions involve small ORFs, which can encode short peptides, in long noncoding RNAs (lncRNAs). Rarely, more dramatic overlap in function has been observed for conventional mRNAs with long open reading frames. The Xenopus VegT mRNA encodes a transcription factor required for endoderm formation in the embryo. The same mRNA also has a structural role in organization of the cytokeratin cytoskeleton. Depletion of VegT mRNA leads to fragmentation of the cytokeratin network in the vegetal cortex of the oocyte. Sequences within much of the mRNA appear to act redundantly in controlling the organization of the cytokeratin network, with a functional element contained within a 300 nt portion of the 3′ UTR sufficient to induce depolymerization of cytokeratin filaments (Ryu, 2015).

A second mRNA with essential coding and noncoding functions is oskar (osk), from Drosophila. Osk protein is expressed specifically at the posterior pole of the oocyte and early embryo, where it is responsible for embryonic body patterning and germ cell formation. In the absence of Osk protein, oogenesis progresses normally except for the failure to assemble posterior pole plasm in the oocyte. Although eggs are produced, the embryos fail to form abdominal segments and die. This coding role for osk places substantial constraints on the mRNA sequence. The open reading frame is constrained by the need to encode Osk protein. In addition, noncoding regions are constrained by the elaborate regulation required to restrict Osk protein expression to a discrete subcellular domain: misexpression of Osk is just as lethal as loss of Osk. The osk mRNA is also needed, independent of its coding role, for progression through oogenesis. In the absence of osk mRNA a variety of defects emerge in the organization of the egg chamber, with oogenesis arrested and no eggs produced ( Jenny, 2006; Kanke, 2015). These defects are present well before the developmental stage when Osk protein first appears, and the osk RNA function does not require the osk coding region. Instead, the osk mRNA 3' UTR mediates the noncoding function, placing constraints on the sequence of that region of the mRNA (Ryu, 2015).

Deployment of Osk protein specifically at the posterior pole of the oocyte involves a complex and coordinated program of mRNA localization and translational control. osk mRNA is transcribed in the nurse cells and transported into the oocyte through cytoplasmic bridges. Within the oocyte, osk mRNA is transiently enriched at different positions, culminating in persistent posterior localization starting at stage 9; this is when Osk protein first accumulates. Translational repression serves to prevent expression from osk mRNA that has not yet been localized, or has failed to become localized. Once osk mRNA is localized, translational activation must then override repression and allow Osk protein to be made. Many factors and regulatory elements are required for this regulation, with most of the elements positioned in the 3'UTR. Among the elements are a number of binding sites for Bru (BREs and others), clustered in two regions of the 3'UTR: the AB region (close to the coding region), and the C region (close to the 3' end). Mutation of all the BREs disrupts translational repression, revealing the role of Bru as a repressor. By contrast, mutation of only the C region cluster of BREs disrupts translational activation, implicating Bru in activation, as well as repressio in the C region also disrupts translational activation (Ryu, 2015).

The noncoding role of osk mRNA is mediated by the 3' UTR. Of greater importance to the noncoding requirement for osk mRNA are sequences positioned close to the mRNA 3' end in the C region, including the Bru binding sites that activate translation. These C region Bru binding sites contribute to sequestration of Bru, but also play a separate and essential role in osk noncoding function. Additional sequences essential for the noncoding function, which do not bind Bru, are positioned nearby. Some of the sequences in this region appear to act by binding poly(A) binding protein (PABP). However, the mutations which most strongly disrupt osk RNA function are not PABP binding sites, and the factor expected to bind them has not been identified (Ryu, 2015 and references therein).

To better understand the roles of the C region of the osk mRNA this study sought proteins which bind specifically to the essential sequences. Bicoid Stability Factor (BSF), a protein previously found to act in stabilizing the bicoid mRNA, binds to the osk C region, with binding dependent on sequences most critical for osk RNA function early in oogenesis. Surprisingly, it was found that the same sequences are also required again, late in oogenesis, for regulation of osk expression. BSF mediates this later function, as shown in two complementary approaches. However, binding of BSF to the C region does not appear to be responsible for the early function, as certain mutations which substantially reduce BSF binding have no effect on the noncoding role of osk mRNA. Why regulatory and functional elements should be superimposed in the RNA sequence is an intriguing question, as the osk 3′ UTR is quite large and thus does not seem to be constrained in size (Ryu, 2015).

Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model

Myotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, a genetic screen was carried using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology (Llamusi, 2013).

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA

The early stages of Drosophila development rely extensively on posttranscriptional forms of gene regulation. Deployment of the anterior body patterning morphogen, the Bicoid protein, requires both localization and translational regulation of the maternal Bicoid mRNA. Evidence is provided that the Bicoid mRNA is also selectively stabilized during oogenesis. A protein, Bicoid stability factor (BSF: CG10302), has been identified and isolated that binds specifically to IV/V RNA, a minimal form of the Bicoid mRNA 3' untranslated region that supports a normal program of mRNA localization during oogenesis. Mutations that disrupt the BSF binding site in IV/V RNA or substantially reduce the level of BSF protein lead to reduction in IV/V RNA levels, indicating a role for BSF in RNA stabilization. The BSF protein is novel and lacks all of the characterized RNA binding motifs. However, BSF does include multiple copies of the PPR motif, whose function is unknown but appears in other proteins with roles in RNA metabolism (Mancebo, 2001).

To search for proteins that may regulate the activity or distribution of BCD mRNA, a focus was placed on the IV/V region, a 271-nt portion of the BCD 3' UTR that supports a normal pattern of mRNA localization during oogenesis. Unlike the complete 3' UTR, the IV/V RNA lacks redundant information for the initial step of BCD mRNA localization. Specifically, the localization activity of IV/V can be greatly reduced by a point mutation (G4496U), while the same mutation has only a subtle and transient effect on the activity of the complete 3' UTR. The absence of functional redundancy is a prerequisite for experiments in which an attempt is made to correlate a protein binding site with a biological activity (Mancebo, 2001).

Extracts prepared from Drosophila ovaries were tested for the presence of proteins that bind to IV/V RNA using a UV cross-linking assay. A number of proteins bind under these conditions. The assays were also performed in the presence of increasing amounts of competitor RNA as an initial test for binding specificity. Most of the bands detected in the assay are unaffected by addition of the competitor, but the binding of four proteins, p55, p70, p80, and BSF, is clearly reduced. To explore a possible role for any of the proteins in BCD mRNA localization, RNA probes corresponding to the isolated parts of IV/V (predicted stem-loops IV and V) were used in separate binding assays; RNAs IV and V have no localization activity in vivo and thus may fail to bind one or more localization factors. Many proteins bind equally well to all probes. Two proteins, p55 and p70, bind to V RNA but not IV RNA, suggesting that they recognize sites contained entirely within V. Finally, p80 and BSF bind much better to IV/V RNA than to either of the isolated parts (which do not support mRNA localization) and are thus the best candidates to act in mRNA localization. To explore further a possible role for the cross-linking proteins in BCD mRNA localization, a binding assay was done with a point-mutated IV/V RNA (G4496U) that interferes with mRNA localization in vivo. The G4496U mutation has no effect on the binding of any of the proteins detected in this assay. Although this result does not rule out involvement of any of the binding proteins in BCD mRNA localization, it does suggest that other roles may be more likely (Mancebo, 2001).

The strategy for testing the role of BSF was to first identify mutations in IV/V RNA that interfere with BSF binding in vitro and then to determine the consequences of these same mutations in vivo. LS mutagenesis was used to create a series of 27 mutants that collectively alter most of the 271 nt of IV/V. Each mutant replaces a 10-nt segment of IV/V with a synthetic sequence. Wild-type and mutant IV/V RNAs were used as probes in binding assays. One mutant RNA, LS15, is most severely impaired in BSF binding. Several others (e.g., LS19 and LS20) are also impaired but to a lesser extent. Almost all of the same LS mutants were also tested in vivo. Each mutant was introduced into a reporter construct, transgenic fly strains were established, and patterns of mRNA localization in transgenic ovaries were monitored by in situ hybridization. For the LS15 mutant, no localized reporter mRNA was detected in any of four independent transgenic fly stocks, and the underlying cause was unique among all mutants tested: the LS15 mutant fails to accumulate any mRNA. In comparison, the LS11 mutant, which is completely defective in mRNA localization, retains normal levels of transgene mRNA. The simplest interpretation of these results is that the LS15 mutant destabilizes the IV/V RNA. The data point to the likelihood that LS15 mRNA is unstable and that instability is the consequence of the defect in BSF binding (Mancebo, 2001).

BSF is a protein of 1,412 amino acids. The most notable feature of the BSF sequence is the presence of seven copies of the PPR motif, with four copies adjacent to one another near the amino terminus and three copies dispersed over the carboxyl-terminal half of the protein. The PPR motif, which is usually about 35 amino acids long, has no assigned function but has already been identified in over 200 proteins that are widely represented in plant organelles. Two proteins that contain the motif have been characterized genetically, and each plays a role in RNA metabolism. Notably, the PPR-containing PET309 protein has been shown to act in either processing or stabilization of certain RNAs. Based on these observations, BSF is a member of a new protein family that may have a common function in RNA metabolism (Mancebo, 2001 and references therein).

Sequence comparisons of BSF with those of the GenBank database identify two proteins that are most closely related, a human leucine-rich protein of unknown function (expectation [E] value of <10^-136) and a predicted Drosophila protein (E <10^-68). Both proteins also contain multiple copies of the PPR motif, although the extensive homology between BSF and the leucine-rich protein is not limited to these repeated structural elements (Mancebo, 2001).

Flies transheterozygous for bsf¹ mutation and the deficiency Df(2L)M36F-S5 are viable and fertile, with no obvious morphological defects in oogenesis. When eggs from such females are fertilized by wild-type or bsf¹ sperm, they progress normally through embryogenesis and display no cuticular pattern defects (development is arrested later for bsf¹/bsf¹ individuals because of the 25E lethal mutation on the chromosome). As a first test for a molecular defect in the ovaries of bsf¹/Df(2L)M36F-S5 females, the level of endogenous BCD mRNA was examined but no substantial difference was found relative to the wild type. However, the apparent mRNA instability phenotype associated with LS15 (the LS mutant defective in BSF binding) is detected for transgenes containing only the IV/V portion of the BCD mRNA 3' UTR, while deletion from the complete BCD 3' UTR of the region corresponding to LS15 has no substantial effect on mRNA levels. Thus, LS15 can block the action of only a single component of a redundant stabilizing system, and reduction of BSF activity would only be detected when redundancy is eliminated. Accordingly, the level of the reporter mRNA bearing the wild-type IV/V RNA in flies transheterozygous for bsf¹ and Df(2L)M36F-S5 was examined. Compared to control flies, the bsf mutant flies display a consistent three- to five-fold reduction in the level of wild-type IV/V RNA. There is no direct evidence that proves a mechanism by which a reduction in BSF levels reduces the level of IV/V RNA. Nevertheless, the fact that BSF binds to sequences within IV/V RNA argues for a posttranscriptional role, an interpretation consistent with the mRNA stabilization role suggested for BSF by the LS mutant analysis (Mancebo, 2001).

The subcellular distribution of BSF protein was determined by immunofluorescent detection in whole-mount ovaries. At all stages of oogenesis the protein is cytoplasmic. During the previtellogenic stages of oogenesis BSF is present in both the nurse cells and the oocyte at similar levels. Within the nurse cells BSF appears primarily in regions surrounding the nuclei, and within these regions the protein is often concentrated in a punctate pattern. As oogenesis proceeds, the tight association of BSF with nurse cell nuclei is lost. The particulate appearance of BSF is enhanced, but the particles are more evenly dispersed throughout the cytoplasm of the nurse cells. In the oocyte the BSF levels are reduced relative to the nurse cells. At no time does BSF appear to be concentrated at sites of BCD mRNA accumulation, at either the apical regions of the nurse cells or the anterior margin of the oocyte (Mancebo, 2001).

The consequence of mutating either the IV/V RNA stability element or bsf is the elimination or reduction, respectively, of the reporter mRNA bearing the IV/V 3' UTR. In contrast, mutation of bsf has no discernable effect on endogenous BCD mRNA. Similarly, deletion of the LS15 region from the full BCD 3' UTR is tolerated, and the deletion mutant RNAs are readily detectable. The striking context dependence of mutating either the cis or trans components of this RNA stabilization system suggests that there is redundancy in the stabilization process: sequences outside IV/V are sufficient for stabilization, and another factor(s) can perform the same function as BSF. This redundancy is not surprising given the redundancy already demonstrated for localization of BCD mRNA. Indeed, the IV/V subdomain of BCD RNA was used in this work because it lacks the mRNA localization redundancy of the full 3' UTR (Mancebo, 2001).

An alternative explanation for the observation that a mutation in the RNA stabilization element leads to a reduction in the level of IV/V RNA while a deletion of the stabilization element from the full BCD 3' UTR appears to have no affect is suggested by known mechanisms of mRNA stabilization. Specifically, binding of the iron response element protein to sequences in the transferrin receptor mRNA 3' UTR blocks endonucleolytic attack and thus stabilizes the mRNA. It is possible that the LS15 mutant disrupts the BSF binding site but not a nearby nucleolytic cleavage site and thus confers instability. In contrast, the larger deletion mutants that do not affect stability of the BCD 3' UTR might eliminate both the protection and cleavage elements, making them resistant to targeted degradation. Distinguishing among these and other possible explanations will require more detailed analysis of the cis-acting elements (Mancebo, 2001).

In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).

In cnc mutant egg chambers, nuclear movement occurs normally. The nucleus remains cortically localized even after its posterior displacement. Since interference with components of the dynactin complex leads to the dissociation of the nucleus from the cortex, it is believed that the dynactin complex is not affected by the loss of cnc function. However, the polarization of the microtubule network is aberrant in stage 10A cnc oocytes. Higher numbers of microtubules accumulate in the posterior region of the oocyte at the expense of the anterior cortical ring, which dominates the microtubule network of wild-type stage-9 to -10A egg chambers. This second microtubule reorganization could either be the cause of or result from the late displacement of the nucleus. In the first case, cnc would be required for a process that stabilizes and maintains the microtubule polarity after stage 8. Prolonged signaling from posterior follicle cells might be necessary to suppress the reestablishment of microtubule organizing centers (MTOCs) at the posterior pole. The reception of such a signal or its transmission to the cytoplasm might be impaired in the absence of cnc function. In this model, the reassembly of MTOCs in posterior regions would lead to the redistribution of free tubulin and consequently weaken anterior MTOCs. The nucleus would subsequently migrate towards these ectopic posterior MTOCs. BCD mRNA also would become mislocalized since it is known to move, like the nucleus, towards the minus ends of microtubules, i.e., towards the MTOCs (Guichet, 2001).

In the other scenario, cnc would be required specifically for oocyte nucleus anchoring at the anterior cortex. Anterior anchoring might be necessary since there is a massive influx of cytoplasm from the nurse cells to the anterior pole of the oocyte during egg chamber growth. If the nucleus is not properly anchored, these transport processes might dislodge the nucleus from the anterior pole. Why would this mispositioning of the nucleus lead to the reorganization of the microtubule network? Such microtubule reorganizations have not been described in other mutant backgrounds where the nucleus does not reach the anterior cortex, such as grk, cni, mago, and DLis-1. It has been shown that the nucleus gets encaged by microtubules when it arrives at the anterior pole in wild-type oocytes, indicating that the anteriorly localized nucleus acquires a microtubule-nucleating activity. This activity might remain associated with the mispositioned nucleus in cnc egg chambers and might subsequently cause the increased microtubule density in the posterior half of the cnc oocytes (Guichet, 2001).

In both scenarios, the mislocalization of OSK mRNA remains somehow enigmatic. OSK should not localize to the same region to which BCD is transported. However, normal OSK transport from the anterior to the posterior might just be blocked by the mispositioned nucleus and its associated microtubules. Thus OSK might be trapped in the vicinity of the ectopic nucleus on its way to the posterior pole (Guichet, 2001).

Targeting the miR-34a/LRPPRC/MDR1 axis collapse the chemoresistance in P53 inactive colorectal cancer

P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. This study demonstrated that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. Yhis study reports a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC (Yang, 2022).

m6A regulator-mediated RNA methylation modification patterns and immune microenvironment infiltration characterization in patients with intracranial aneurysms

The role of epigenetic modulation in immunity is receiving increased recognition-particularly in the context of RNA N6-methyladenosine (m6A) modifications. Nevertheless, it is still uncertain whether m6A methylation plays a role in the onset and progression of intracranial aneurysms (IAs). This study aimed to establish the function of m6A RNA methylation in IA, as well as its correlation with the immunological microenvironment. This study included a total of 97 samples (64 IA, 33 normal) in the training set and 60 samples (44 IA, 16 normal) in the validation set to systematically assess the pattern of RNA modifications mediated by 22 m6A regulators. The effects of m6A modifications on immune microenvironment features, i.e., immune response gene sets, human leukocyte antigen (HLA) genes, and infiltrating immune cells were explored. Lasso, machine learning, and logistic regression were used for the purpose of identifying an m6A regulator gene signature of IA with external data validation. For the unsupervised clustering analysis of m6A modification patterns in IA, consensus clustering methods were employed. Enrichment analysis was used to assess immune response activity along with other functional pathways. The m6A methylation markers were identified based on a protein-protein interaction network and weighted gene co-expression network analysis. This study identified an m6A regulator signature of IGFBP2, IGFBP1, IGF2BP2, YTHDF3, ALKBH5, RBM15B, LRPPRC, and ELAVL1, which could easily distinguish individuals with IA from healthy individuals. Unsupervised clustering revealed three m6A modification patterns. Gene enrichment analysis illustrated that the tight junction, p53 pathway, and NOTCH signaling pathway varied significantly in m6A modifier patterns. In addition, the three m6A modification patterns showed significant differences in m6A regulator expression, immune microenvironment, and bio-functional pathways. Furthermore, macrophages, activated T cells, and other immune cells were strongly correlated with m6A regulators. Eight m6A indicators were discovered-each with a statistically significant correlation with IA-suggesting their potential as prognostic biological markers. This study demonstrates that m6A RNA methylation and the immunological microenvironment are both intricately correlated with the onset and progression of IA. The novel insight into patterns of m6A modification offers a foundation for the development of innovative treatment approaches for IA (Maimaiti, 2022).

RNA Chemical Proteomics Reveals the N6-Methyladenosine (m6A)-Regulated Protein-RNA

Epitranscriptomic RNA modifications can regulate mRNA function; however, there is a major gap in understanding of the biochemical mechanisms mediating their effects. This study develop a chemical proteomics approach relying upon photo-cross-linking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic RNA. In addition to identifying YTH domain-containing proteins and ALKBH5, known interactors of this modification, this study found that FMR1 and LRPPRC, two proteins associated with human disease, "read" this modification. Surprisingly, this study also found that m6A disrupts RNA binding by the stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42. This work provides a general strategy for interrogating the interactome of RNA modifications and reveals the biochemical mechanisms underlying m6A function in the cell (Arguello, 2017).

Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation

Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. The mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as Bicoid stability factor) have been shown to be necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. This study reports characterization of DmLRPPRC2 (CG14786), a second fruit fly homolog of LRPPRC, and shows that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (CG3021/DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation (Baggio, 2014).

Search PubMed for articles about Drosophila Bif or Rpprc1

Arguello, A. R., DeLiberto, A. N. and Kleiner, R. E. (2017). RNA Chemical Proteomics Reveals the N6-Methyladenosine (m6A)-Regulated Protein-RNA. J Am Chem Soc 139(48):17249-17252. PubMed ID: 29140688

Baggio, F., Bratic, A., Mourier, A., Kauppila, T. E., Tain, L. S., Kukat, C., Habermann, B., Partridge, L. and Larsson, N. G. (2014). Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation. Nucleic Acids Res 42(22): 13920-38. PubMed ID: 25428350

Llamusi, B., Bargiela, A., Fernandez-Costa, J. M., Garcia-Lopez, A., Klima, R., Feiguin, F. and Artero, R. (2013). Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model. Dis Model Mech 6(1): 184-196. PubMed ID: 23118342

Maimaiti, A., Turhon, M., Cheng, X., Su, R., Kadeer, K., Axier, A., Ailaiti, D., Aili, Y., Abudusalamu, R., Kuerban, A., Wang, Z. and Aisha, M. (2022). m6A regulator-mediated RNA methylation modification patterns and immune microenvironment infiltration characterization in patients with intracranial aneurysms. Front Neurol 13: 889141. PubMed ID: 35989938

Mancebo, R., et al. (2001). BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA. Mol. Cell. Bio. 21: 3462-3471. 11313472

Matsushima, Y., Hirofuji, Y., Aihara, M., Yue, S., Uchiumi, T., Kaguni, L. S. and Kang, D. (2017). Drosophila protease ClpXP specifically degrades DmLRPPRC1 controlling mitochondrial mRNA and translation. Sci Rep 7(1): 8315. PubMed ID: 28814717

Yang, Y., Yuan, H., Zhao, L., Guo, S., Hu, S., Tian, M., Nie, Y., Yu, J., Zhou, C., Niu, J., Wang, G. and Song, Y. (2022). Targeting the miR-34a/LRPPRC/MDR1 axis collapse the chemoresistance in P53 inactive colorectal cancer. Cell Death Differ 29(11): 2177-2189. PubMed ID: 35484333

date revised: 5 August 2023

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