Rm62: Biological Overview | References
Gene name - Rm62
Cytological map position - 83D1-83D2
Function - enzyme
Symbol - Rm62
FlyBase ID: FBgn0003261
Genetic map position - chr3R:1,826,143-1,834,028
Classification - DEAD box helicase family
Cellular location - nuclear
|Recent literature||Fear, J. M., Arbeitman, M. N., Salomon, M. P., Dalton, J. E., Tower, J., Nuzhdin, S. V. and McIntyre, L. M. (2015). The Wright stuff: Reimagining path analysis reveals novel components of the sex determination hierarchy in Drosophila melanogaster. BMC Syst Biol 9: 53. PubMed ID: 26335107
This study used a structural equation modeling approach, leveraging natural genetic variation from two studies on Drosophila female head tissues to expand understanding of the sex hierarchy gene regulatory network (GRN). The GRN was expanded adding novel links among genes, including a link from fruitless (fru) to Sex-lethal (Sxl). This link is further supported by the presence of fru binding sites in the Sxl locus. 754 candidate genes were added to the pathway, including the splicing factors male-specific lethal 2 and Rm62 as downstream targets of Sxl. Independent studies of doublesex and transformer mutants support evidence for a link between the sex hierarchy and metabolism, via Insulin-like receptor. The genes added in one population were enriched for genes with sex-biased splicing and components of the spliceosome. Using natural alleles this approach not only identifies novel relationships, but using supervised approaches can order genes into a regulatory hierarchy.
DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, an evolutionarily conserved and interferon-independent role was identified for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhances RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), this study shows that DDX17 binds the stem loops of host precursor-miRNA (pri-miRNA) to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements (Moy, 2014).
RNA helicases control nearly every facet of RNA metabolism, including transcription, splicing, miRNA biogenesis, translation, and decay. Comprising the largest family of helicases, the DEAD-box proteins are found in all three kingdoms of life and share 12 conserved motifs, including the DEAD motif characterized by the amino acids Asp-Glu-Ala-Asp. Although DEAD-box proteins are most appreciated for their roles in RNA metabolism, some have important functions in antiviral defense. For example, mammalian retinoic acid-inducible gene 1 (RIG-I/DDX58) and myeloma differentiation-associated factor 5 (MDA-5), collectively termed RIG-I-like receptors (RLRs), recognize non-self-elements in viral RNAs such as double-stranded RNA (dsRNA) and 5'-triphosphorylated RNA, leading to the transcriptional induction of Type I interferon (IFN-I) and proinflammatory cytokines (Loo, 2011). However, some viruses are not restricted by RLRs in some contexts or encode potent RLR antagonists, and thus additional sensors may have evolved (Moy, 2014).
Although RLRs are not strictly conserved in invertebrates such as mosquitoes and Drosophila, insects use a related helicase to combat viral infection. The DEAD-box helicase Dicer-2 (Dcr-2) is a core component of the RNAi pathway that recognizes double-stranded or structured viral RNAs and cleaves them into 21 nt small-interfering RNAs (siRNAs) (Ding, 2007; Sabin, 2013). Virus-derived siRNAs are loaded into an Argonaute-2 (Ago2)-containing RNA-induced silencing complex that cleaves viral RNA. Additionally, during Drosophila C virus (DCV) infection, Dcr-2 controls induction of the antiviral gene Vago (Deddouche, 2008; Moy, 2014 and references therein).
More recently, several other DEAD-box proteins have been implicated in sensing viral nucleic acids or regulating downstream signaling. For example, DDX41 recognizes intracellular DNA and bacterial cyclic dinucleotides, whereas a complex of DDX1, DDX21, and DHX36 senses viral dsRNA specifically in dendritic cells. Other recently identified helicase sensors or components of antiviral signaling pathways include DDX3, DHX9, and DDX60. Thus, the landscape of DEAD-box helicases in innate immunity is more diverse than previously appreciated, and many antiviral helicases likely remain obscure (Moy, 2014).
As many aspects of innate immunity are conserved in flies as well as many DEAD-box helicases, an RNAi screen was performed to identify novel antiviral helicases. Focus was placed on the arthropod-borne virus (arbovirus) Rift Valley fever virus (RVFV), a tri-segmented negative-sense RNA virus in the bunyavirus family. In humans, RVFV infection typically causes an acute febrile illness but can progress to more severe manifestations such as encephalitis and hemorrhagic fever with 1%–3% mortality. In livestock, infection is particularly lethal with 100% abortion rates and near 100% fatality in neonates. No effective vaccines or therapeutics exist for RVFV infection, and therefore additional targets for pharmacologic intervention are needed. Furthermore, it has been shown that RVFV is not restricted by RLRs in some contexts including fibroblasts, suggesting that other sensors may restrict this pathogen (Moy, 2014).
This study identifies Drosophila Rm62 as a novel host factor that restricts RVFV infection in vitro and in vivo. This restriction was specific for bunyaviruses, as Rm62 also controlled the replication of the distantly related bunyavirus La Crosse virus (LACV), but not viruses from the three other families tested. Remarkably, the antiviral role of Rm62 was conserved in human cells, as the human homolog DDX17 restricted RVFV infection. DDX17 was identified in a high-molecular-weight complex with Drosha and later shown to regulate the Microprocessor complex that mediates pri-miRNA processing and miRNA biogenesis, but its direct RNA targets are not fully known. Using CLIP-seq, this study found that in addition to binding cellular RNAs, DDX17 also interacts with RVFV RNA, likely via structured viral RNA elements. Striking similarities were found in the mode of recognition for host and viral RNA: DDX17 binds a subset of pri-miRNA hairpins along with a well-characterized hairpin on the RVFV genome. Cloning this hairpin into Sindbis virus (SINV) decreased its replication in a DDX17-dependent manner in human and insect cells, indicating a direct antiviral function for DDX17 binding to viral RNA. Taken together, these data expand the understanding of DDX17 recognition of cellular and viral RNAs as well as the scope of DEAD-box helicases in antiviral immunity, demonstrating that the immune functions of DEAD-box genes can be evolutionarily conserved from insects to humans (Moy, 2014).
Emerging data have begun to uncover specialized functions for mammalian DEAD-box helicases in immunity, particularly in the sensing of viral nucleic acids to activate interferon induction (Fullam, 2013). However, whether DEAD-box helicases play interferon-independent roles in antiviral immunity remains unclear. Moreover, whether antiviral DEAD-box helicases exist primarily in mammals or evolved immune functions in lower organisms has not been fully explored. This study has discovered a specific and evolutionarily conserved role for the helicase DDX17 in restricting infection with RVFV, a major human arbovirus that lacks effective therapeutics (Moy, 2014).
Through an RNAi screen in Drosophila cells, Rm62 was identified as an anti-RVFV helicase gene. Rm62 is also essential for resistance to RVFV infection in vivo, as RVFV-challenged Rm62-deficient flies showed increased viral replication and mortality. Loss of Rm62 increased LACV infection but not SINV, VSV, or DCV infection, and silencing closely related DEAD-box helicase genes had no effect on RVFV. Thus, Rm62 is as an essential and specific virus restriction factor in flies (Moy, 2014).
Mammals encode two orthologs of Rm62, DDX5 and DDX17, which have been widely studied in transcriptional coactivation, mRNA splicing, and miRNA processing. In addition, previous studies have implicated DDX5 and DDX17 in promoting the replication of several viruses, such as hepatitis C virus and influenza virus. This study found that silencing DDX17 but not DDX5 increased RVFV replication in human U2OS cells, whereas DDX17 overexpression inhibited RVFV infection. Although a antiviral function for DDX5 cannot be ruled out, as DDX5 depletion upregulated DDX17 expression and it was not possible to efficiently knock down both proteins simultaneously in U2OS cells, basal DDX5 levels were not able to compensate for DDX17 loss. Therefore, the antiviral activity of DDX17 is evolutionarily conserved from invertebrates to mammals, suggesting an ancient origin for DEAD-box helicases in innate immunity. DDX17 joins a growing list of antimicrobial DEAD-box proteins that function as cytoplasmic sensors for viral nucleic acids. For example, DDX41 binds DNA to control IFN-I and proinflammatory cytokine induction (Zhang, 2011). Additionally, DDX3 interacts with IKKε and TBK-1 to regulate IFN-I activation downstream of virus recognitio. In contrast, DDX17 is dispensable for antiviral gene expression, suggesting that DDX17 acts independently of IFN-I, which is distinct from previously defined antiviral DEAD-box genes (Moy, 2014).
Previous reports have proposed that Rm62 and DDX17 regulate RNAi, which is a well-characterized antiviral pathway in invertebrates. In Drosophila cells, Rm62 has been shown to bind Ago2 and control siRNA-mediated silencing (Ishizuka, 2002). Based on these findings, one study suggested that Rm62-deficient flies infected with Drosophila X virus (DXV) have increased mortality due to defective antiviral RNAi (Zambon, 2006); however, this study did not use sibling-matched or uninfected controls and did not monitor viral replication. In mammalian cells, DDX5 and DDX17 are found in the Microprocessor complex and regulate miRNA biogenesis. However, the current data suggest that Rm62 and DDX17 restrict viral infection in an RNAi-independent manner. First, it was found that Rm62 controls the replication of RVFV but not VSV, SINV, or DCV, viruses that are restricted by antiviral RNAi in flies. Second, Rm62 has not been found to control siRNA- or miRNA-mediated RNA silencing in more recent in vitro and in vivo screens, which was confirmed in current experiments. Third, RNAi is not generally thought to restrict viral infection in mammalian somatic cells, whereas its antiviral function in embryonic and undifferentiated mammalian cell types may be active. Lastly, depletion of the Microprocessor component Drosha, which can act as an interferon-independent antiviral factor, did not impact RVFV infection. Although there may be additional complexity and interplay between DDX17, miRNA biology, and antiviral defense, the current data suggest that DDX17’s antiviral function is independent of its role in miRNA biogenesis (Moy, 2014).
LIP-seq studies revealed that DDX17 physically associates with viral RNA to control viral infection. One of the DDX17-binding peaks, corresponding to the IGR between the N and NSs genes on the genomic S segment, efficiently precipitated DDX17 from cell lysates. Interestingly, this region forms an extensive hairpin (Sabin, 2013), suggesting that DDX17 may recognize highly structured stem loops on viral RNAs. Furthermore, in infected cells, DDX17 forms cytoplasmic puncta overlapping with RVFV N. This relocalization to viral replication complexes may allow DDX17 to access and bind structured viral RNA elements and thereby limit replication (Moy, 2014).
Indeed, by expressing the RVFV hairpin from SINV (SINV-hp), it was demonstrated that SINV becomes hypersensitive to DDX17 in both human and insect cells. This suggests that binding of DDX17 to viral RNA is sufficient to mediate its antiviral effect. How this binding limits viral replication remains to be clarified in future studies. DDX17 may associate with additional protein cofactors that mediate its antiviral function. For instance, DDX17 has been shown to bind Dcp2 and Dcp1a, which remove the 5' cap from mRNAs, and the exonuclease Xrn1, which mediates 5'-to-3' RNA degradation. Interestingly, a recent study showed that Drosophila Dcp2 restricts RVFV infection, although this may be in an indirect manner by limiting the pool of cellular mRNA substrates that RVFV utilizes for its own replication. DDX5 and DDX17 also bind components of the RNA exosome, a complex that catalyzes 3'-to-5' RNA degradation. Consequently, DDX17 may act as the sensor that brings viral RNA targets to the RNA degradation machinery, or it may unwind viral RNAs to facilitate degradation. The decapping machinery, exosome, and DDX17 have additionally been linked to the antiviral protein ZAP. ZAP is known to restrict SINV replication in human cells, and this study found a modest effect of DDX17 depletion on WT SINV, albeit this effect was not as strong as with SINV-hp. In contrast, flies do not encode a ZAP homolog, and Rm62 silencing had no impact on WT SINV replication in insect cells. As maximal DDX17 restriction of SINV depended on the RVFV stem loop in both cells types, DDX17's antiviral function in RVFV infection is likely independent of ZAP and dependent on direct viral RNA binding (Moy, 2014).
Why DDX17 specifically targets bunyaviral RNAs is also uncertain, as the rules that direct RNA-binding activity of DDX17, as well as DEAD-box proteins in general, are difficult to decipher. It is suspected that antiviral specificity derives from a combination of cellular localization and specific RNA structures. The data suggest that SINV and VSV do not have the appropriate targeting signals. In addition, the correlation between DDX17 expression pattern and viral pathogenesis must be characterized. DDX17 is ubiquitously expressed and is not transcriptionally induced by IFN-I, and the data suggest that subcellular localization but not expression level is responsive to infection. Further studies will a better definition of the regulation of DDX17 and explore the relationship between tissue type and antiviral activity (Moy, 2014).
Beyond elucidating new DDX17 functions in immunity, the data reveal important insights into the mechanism of DDX17 recognition for diverse RNAs. The data suggest that DEAD-box proteins are highly amenable to CLIP-seq analysis. This study found that DDX17 cellular mRNA targets are enriched for CT- and CA-repeat elements, suggesting that primary sequence contributes to mRNA recognition. In contrast, DDX17-bound pri-miRNAs were not enriched for this element or any other linear sequence; instead, DDX17 was localized to the miRNA stem, suggesting that it recognizes pri-miRNAs via secondary structure. Bound pri-miRNAs were compared with two other studies that monitored miRNAs upon loss of DDX17. One identified 94 miRNAs that were decreased upon loss of p72 in mouse embryonic fibroblasts that derive from 82 pre-miRNAs expressed in U2OS cells, of which 32% were directly bound by DDX17 in the current studies. Another study identified 317 miRNAs misregulated by DDX17 depletion in HaCaT cells; of the 160 DDX17-bound miRNAs from the current study, 60 were analyzed in their cells, and 30 were found to be regulated by DDX17 (50%). Furthermore, the first study identified a sequence motif in the 3' flanking segment of a subset of pri-miRNAs that were impacted by DDX17 levels ([GTA]CATC[CTA]), and focus was placed on miR-21, a miRNA that the current also identified as bound by DDX17. The previous study demonstrated by in vitro binding assays that both the motif as well as a complete stem loop were required for full binding activity. Altogether, these data suggest that DDX17 recognizes the pri-miRNA stem in the context of a 3' tail. This would bias DDX17 binding to pri-miRNAs over pre-miRNAs because some additional binding energy would be derived from the flanking regions and would facilitate DDX17 binding to stem loops within larger RNAs as is found in viral RNAs. In further support of this, the ([GTA]CATC[CTA]) sequence motif was over-represented in mRNA peaks of the current study, suggesting that this sequence is indeed a preferred binding site for DDX17 in diverse RNAs (Moy, 2014).
In conclusion, these data reveal striking parallels between DDX17 recognition of pri-miRNAs and viral RNAs: in both cases, DDX17 targets a structured stem loop, either to facilitate miRNA processing or to mediate virus inhibition (Moy, 2014).
Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. This study shows that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase Su(var)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of Su(var)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range (Boeke, 2011).
This study has identified Rm62 as an interactor with the N-terminus of Su(var)3-9. Interestingly, this interaction domain is shared between the Su(var)3-9 and eIF2γ and could therefore mediate the interaction between Rm62 and both proteins. This study focused on the analysis of the nuclear interaction of Rm62 and Su(var)3-9 as it seems to be important for the efficient shut down of highly activated genes such the hsp70. In accordance with the previously described role of histone modifications at the heat shock locus, a strong H3K9 methylation was observed at the hsp70 gene before heat shock activation that disappears after heat shock and slowly reappears when cells recover from heat shock. This methylation is highly dependent on the presence of Rm62 as it is strongly reduced in Rm62 mutant fly strains. Rm62 mutation not only leads to less H3K9 methylation at the heat shock loci but also leads to a global reduction of the H3K9me2 mark in euchromatin. This suggests a widespread mechanism of methyltransferase recruitment mediated by the interaction between Rm62 and Su(var)3-9 (Boeke, 2011).
Histone modifications play a crucial role in regulating gene expression. The hsp70 locus provides an excellent model promoter for rapidly switching between the on and the off state of transcription and it has been shown to be regulated at multiple levels including histone modification. One of the factors that get recruited to the heat shock promoter immediately after activation is the Rm62, which this study identifies as an interactor with the histone methyltransferase Su(var)3-9. Despite being recruited immediately after heat shock, Rm62 plays a role in transcriptional shut down after removal of the heat shock (Buszczak, 2006). It has been suggested that the RNA helicase activity is required for the efficient removal of the RNA from its site of transcription, which in turn is important for the resilencing of the gene (Buszczak, 2006). However, a more direct role in the generation of the repressed state could not be excluded. Since a strong, Rm62 dependent recruitment of Su(var)3-9 to the promoter was observed after heat shock that is important for the reestablishment of H3K9 methylated chromatin, it is proposed that the interaction between the two proteins contributes to the regeneration of a repressive chromatin structure after heat shock. Buszczak observed a prolonged phosphorylation of H3S10 at the hsp70 locus in flies that carry a mutation in Rm62 that may very well be due to a failure of recruiting Su(var)3-9 and H3K9 methylation in absence of Rm62. The phosphorylation of H3S10 is severely impaired when the neighboring residue (H3K9) is methylated by Su(var)3-9 in vitro. The recruitment of a H3K9 methyltransferase to the hsp70 gene after heat shock may therefore prevent an efficient phosphorylation of H3S10 thereby favoring the reestablishment of a repressed chromatin structure. At the same time could the increased recruitment of a H3S10 kinase prevent a premature methylation of K9 via the recruited methyltransferases, which may explain the striking kinetic difference observed between the binding of Su(var)3-9 and the accumulation of H3K9 methylated histones. These findings may therefore provide another example of a phospho-methyl switch where a strong interdependence of histone methylation and histone phosphorylation is observed on adjacent residues. Alternatively, the lack of histone methylation after heat shock that is seen by immunofluorescence and by ChIP could be due to the fact that histones are completely removed after heat shock and are only reassembled during recovery. In this case the recruitment of Su(var)3-9 would lead to an increased local concentration of the methyltransferase at the site of the promoter, which could (re-)methylate the ejected histones leading to the regeneration of a repressed state after heat shock. This may in fact also explain the seemingly paradoxical effect of HP1 localisation at heat shock puffs. The binding data could also suggest that the recruitment of Su(var)3-9 is in fact important for gene activation, since it is found to bind to the promoter immediately after the induction of transcription. However, this is unlikely, since an effect of Su(var)3-9 and Rm62 removal is observed on the shut down of hsp70 transcription but not on it's induction (Boeke, 2011).
An alternative explanation for the apparent discrepancy between Su(var)3-9 binding and H3K9 methylation could be the need for an additional signal for the enzyme to become active. Such a signal could be an external signal such as a posttranslational modification or an internal signal such as the RNA transcribed from the hsp70 locus itself. Immediately after heat shock a short burst of small RNAs can be detected that are released from the heat shock locus. Considering the fact that Rm62 also plays a role in RNAi mediated silencing (Csink, 2006), this pulse of small RNAs might in fact be the cause for the heat shock dependent recruitment of Rm62 to the hsp70 locus that this study observed. The data suggest that Su(var)3-9 is then recruited to the hsp70 locus via protein-protein interactions where it methylates the histones that are assembled onto the promoter during repression. However, the possibility was not tested that the RNA stimulates the activity of Su(var)3-9, which could also contribute to the delayed histone methylation (Boeke, 2011).
Finally it cannot be excluded that, in addition to Su(var)3-9, a demethylase is recruited to the hsp70 locus, which removes the histone methylation from the promoter bound histones. Indeed, the jmjC family member dUTX, which contains a H3K27 specific demthylase associates with the elongating RNA polII enzyme and is recruited to the hsp70 locus after heat shock. It is very likely that multiple redundant mechanisms play a role in the re-silencing of the hsp70 genes after heat shock with all the possibilities discussed above being involved. In light of the novel finding of a functional interaction between Su(var)3-9 and Rm62 it will be interesting to investigate whether this interaction my also provide a mechanistic link between the shut down of highly active genes and the silencing of repetitive DNA elements via the generation of short non translated transcripts that may help in recruiting a histone methyltransferase. Similar mechanisms have been shown to operate in S. pombe but were so far not identified in higher eukaryotes (Boeke, 2011).
Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, this study identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex is unlike previously described RNA-induced silencing complexes and associates with the DEAD-box helicase RM62, a protein previously implicated in RNA silencing. In flies, Blanks is highly expressed in testes tissues and is necessary for postmeiotic spermiogenesis, but loss of Blanks is not accompanied by detectable transposon derepression. Instead, genes related to innate immunity pathways are up-regulated in blanks mutant testes. These results reveal Blanks to be a unique component of a nuclear siRNA/dsRNA-binding complex that contributes to essential RNA silencing-related pathways in the male germ line (Gerbasi, 2011).
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder seen in Fragile X premutation carriers. Previous studies found that Fragile X rCGG repeats are sufficient to cause neurodegeneration and that the rCGG repeat-binding proteins Pur alpha and hnRNP A2/B1 can modulate rCGG-mediated neuronal toxicity. To explore the role of Pur alpha in rCGG-mediated neurodegeneration further, a proteomic approach was taken, and more than 100 proteins were identified that interact with Pur alpha. Of particular interest is Rm62, the Drosophila ortholog of p68 RNA helicase, which could modulate rCGG-mediated neurodegeneration. This study shows that rCGG repeats decreased the expression of Rm62 posttranscriptionally, leading to the nuclear accumulation of Hsp70 transcript, as well as additional mRNAs involved in stress and immune responses. Together these findings suggest that abnormal nuclear accumulation of these mRNAs, likely as a result of impaired nuclear export, could contribute to FXTAS pathogenesis (Qurashi, 2011).
Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. An HMGB-like protein, DSP1 (Dorsal Switch Protein 1), has been classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. This study identified a DEAD-box RNA helicase, Rm62, as partner of DSP1 in a 250-kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3- to 12-h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. This study shows that an RNA helicase is involved in the maintenance of homeotic genes (Lamiable, 2010).
Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and this study describes the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP's translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo (Cziko, 2009).
It is suggested, that as for previously identified sev-dfmr1 suppressors Ago1, Lgl, and Me31b, analysis of PABP, Smd3, Rm62, Orb2, and Dco proteins, encoded by the sev-dfmr1 suppressor genes identified in this study, will help elucidate how dFMR1 works in translational regulation, RNA targeting and localization, and ncRNA pathway function (Cziko, 2009).
Three lines of evidence indicate that the genes identified encode proteins with translational repressor activity. First, with the exception of Dco, all of these proteins have been previously implicated in some aspect of RNA metabolism and are present on dFMR1-containing neuritic granules in which RNA is repressed and transported. Second, the rough-eye phenotype observed in sev-dfmr1 has been linked to the ability of FMRP to repress mRNA translation. Thus, it would be expected that the phenotype would be alleviated by mutations that reduce the efficiency of translational repression. Third, overexpression of Dco, Pabp, Orb2, or Rm62 inhibits the dendritic growth of neurons, a phenotype predicted for neuronal translational repressors. These observations are consistent with the idea that translation of RNAs in neurites, which promotes dendritic branching, is inhibited by overexpression of Dco, Pabp, Orb2, or Rm62. Thus, genetic interaction data, molecular localization, and one functional test in dendrites indicate that Dco/Dbt, PABP, Rm62, or SmD3 function as neuronal translational repressors (Cziko, 2009).
The identification of several canonical translational-factor encoding genes as suppressors of sev-dfmr1 highlights the point that individual translational control molecules work in multicomponent complexes and therefore have several functional interactions. PABP is one example of a protein that is currently believed to perform two opposing functions of translational control. In addition to its well-studied role as a translational activator, PABP can mediate translational repression, e.g., of Vasopressin mRNA although the exact mechanism remains unclear. Dual roles in activation and repression are also suggested by the observation that reduced or elevated levels of PABP have similar effects at the Drosophila neuromuscular junction (NMJ). Additionally, PABP associates with particles containing BC1, a neuron-specific noncoding RNA with translational repressor function, as well as a CYFIP-FMRP complex that may function as a repressor in some contexts but as an activator in others. Similarly, Orb2 homologs (CPEBs) though required for translational activation of CPE-containing mRNAs via poly-A polymerase, also allow translational repression in combination with Maskin or Cup proteins (Cziko, 2009).
It was somewhat surprising that SmD3, a splicing factor, was identified in a screen for translational repressors. However, SmD3 has additional nonsplicing functions: in Caenorhabditis elegans, the Sm proteins are required for germ cell mRNP assembly and RNA localization. Such a role in translational regulation and mRNP assembly is more consistent with functions predicted by the genetic experiments (Cziko, 2009).
Rm62/Dmp68 is a member of the DEAD-box helicase family that has been shown to be associated with a dFMR1-containing RNAi silencing complex. It also has additional roles during transcription and mRNA processing as well as potentially in miRNA processing as part of the Drosha complex. Based on the biochemical evidence for Rm62's presence in FMRP-containing complexes, it is not surprising that rm62 mutations show strong genetic interactions with dfmr1. However, the mechanism of suppression remains unknown (Cziko, 2009).
Finally Dco/Dbt, is by far the most elusive protein in regard to its potential function in the translational regulatory pathway. Dco/Dbt, a casein kinase I (CKI) is best known from circadian biology where it phosphorylates Per and expedites its degradation. dFMR1 protein has several phosphorylation sites, one of which in S2 cells has been demonstrated to be phosphorylated by a CKII protein. While the functional requirement for CKI-dependent dFMR1 phosphorylation is as of yet not understood, there is considerable evidence that the phosphorylation state of FMRP may actually determine its role in translation. Biochemical data demonstrate that most FMRP in granules is in the phosphorylated state while FMRP in the polysome fraction is dephosphorylated, suggesting a mechanism to switch state from an activator to a repressor, and an important regulatory role for kinases that phosphorylate FMRP (Cziko, 2009).
Another interesting potential link between the two proteins is the behavioral observation that patients with Fragile-X Mental Retardation often display circadian disturbances. This altered circadian rhythm is also present in the Drosophila dfmr1 mutants that usefully model fragile-X syndrome (Cziko, 2009).
The identification of these proteins as sev-dfmr1 modifiers illustrates the many possibly regulatory roles of RNA-associated proteins. In addition, the data associating Dco/Dbt with RNA regulation indicates unexplored and novel mechanisms of RNA regulation in neurons (Cziko, 2009).
Given that dFMR1/FMRP is thought to function in miRNA-dependent translational repression, it was of particular interest to asking whether these dFMR1 interactors had any role in this pathway. To address this issue, a sensitive in vivo assay that uses a fluorescent reporter was employed to reveal the strength of translational repression via an endogenous (bantam) miRNA. When combined with genetic mosaic analysis, this assay can be used to study null mutations in candidate genes, as long as the mutations do not cause cell lethality. The assay appears more sensitive than typically used cell-based assays on the evidence of prior analysis of Me31B, whose requirement for miRNA function, clearly seen in the in vivo assay, is only evident in double-knockdown experiments in the more commonly used cell-culture assays (Cziko, 2009).
In vivo experiments revealed no requirement for the sev-dfmr1 interacting proteins Dco, Orb2, Rm62, and SmD3 in miRNA repression. For reasons explained above, it is unlikely that this reflects a weakness in the experimental assay for miRNA function. A bigger surprise was the finding that the dFMR1 itself appeared dispensable for miRNA function in vivo. Because the allele used is a well-characterized null allele, and the absence of dFMR1 in the mutant clones is confirmed by antibody staining, the conclusion that dFMR1 is not a core, essential component of the RISC/miRNA pathway is strong. This conclusion is not inconsistent with any of the existing data showing biochemical association between RISC and FMRP and genetic interactions between Ago1 and FMRP. However, it is also consistent with recent observations indicating the dispensability of FMRP for RISC function in cultured cells. It is suggested that the function of dFMR1 and, by extension, FMRP may be restricted to a subset of transcripts, for instance those with UTRs containing both FMRP binding motifs and miRNA target elements. Indeed similar models that account for the mRNA specificity of FMRP have been previously proposed (Cziko, 2009).
These data provide a foundation on which to design further experiments to understand the specific roles of FMR1 and its interacting proteins in translational control (Cziko, 2009).
RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. Evidence is presented for a functional relationship between RNAi and the gypsy insulator of Drosophila. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression (Lei, 2006).
These results suggest the existence of an RNA species required for the formation or integrity of insulator bodies, perhaps a product of processing by Argonautes and the other RNAi machinery. The putative RNA helicase Rm62 may be recruited to insulator complexes through physical interaction with CP190 and RNA. Although it is unknown at what mechanistic step Rm62 acts in RNAi, Rm62 may act downstream of Argonautes to unwind or remodel RNA-insulator protein complexes, thereby disrupting gypsy insulator activity and nuclear organization. Proper insulator body localization requires an intact nuclear matrix, and early observations identified RNA as an important component of this nuclear scaffold. Future studies should determine the identity of putative gypsy insulator associated RNAs. These results suggest a previously unknown function of the RNAi machinery in the control of nuclear architecture to effect changes in gene expression (Lei, 2006).
Terminating a gene's activity requires that pre-existing transcripts be matured or destroyed and that the local chromatin structure be returned to an inactive configuration. This study shows that the Drosophila homolog of the mammalian P68 RNA helicase plays a novel role in RNA export and gene deactivation. p68 mutations phenotypically resemble mutations in small bristles (sbr), the Drosophila homolog of the human mRNA export factor NXF1. Full-length hsp70 mRNA accumulates in the nucleus near its sites of transcription following heat shock of p68 homozygotes, and hsp70 gene shutdown is delayed. Unstressed mutant larvae show similar defects in transcript accumulation and gene repression at diverse loci. p68 mutations were found to be allelic to Lighten-up, a known suppressor of position effect variegation. These observations reveal a strong connection between transcript clearance and gene repression. P68 may be needed to rapidly remove transcripts from a gene before its activity can be shut down and its chromatin reset to an inactive state (Buszczak, 2006).
p68 mutant animals turn off genes slowly and incompletely. These include the developmentally regulated early ecdysone response genes E74 and E75, rDNA genes within the nucleolus, and many other active genes that normally cease transcription following heat shock, and the heat-shock response genes themselves during recovery from stress. This prolonged gene activity in p68 mutant animals appears to be normal in regard to chromatin organization, RNA production, and RNA processing. For example, long after hsp70 genes have shut off in wild-type animals, hsp70 genes in p68 mutants retain high levels of H3phosS10 modification, abundant HSF transcription factor, and active RNA polymerase II, and they continue to incorporate Br-UTP into RNA (Buszczak, 2006).
These experiments rule out several potential explanations for the continuing gene activity in p68 mutant animals. First, mammalian P68 has been proposed to facilitate splicing by unwinding base-pairing between the U1 RNA and the 5'-splice junction. Thus, effects on gene shutdown might be secondary to missplicing of direct shutdown regulators. However, no evidence was found that Drosophila P68 affects splicing. The level of unspliced hsp83 transcripts did not increase beyond a small rise attributable to ongoing transcription, nor could unspliced E74 or E75 gene transcripts be detected in p68 mutants. Genes lacking introns such as hsp70 were affected in the same manner as intron-containing genes. Second, mammalian P68 has been implicated as a direct transcriptional regulator, and may participate in activating p53 target genes (Bates. 2005). However, Drosophila P68 seems unlikely to act as a transcriptional activator, because the affected genes were diverse and unrelated in function (including the nucleolus), and were up-regulated rather than down-regulated (Buszczak, 2006).
A final possibility is that secondary affects of p68 on nuclear physiology feed back and arrest gene shutdown. For example, the delay in HSP70 production caused by p68 mutation might slow the activation of the negative feedback loop by which chaperone proteins including HSP70, HSP90, and HSP40 are proposed to normally shut down heat-shock-induced gene expression after removal from stress. However, it seems highly unlikely that the diverse genes whose shutdown depends on p68 function, including rDNA genes, ecdysone response genes, and the cellular genes repressed by heat shock, are all subject to such feedback regulation. Instead, the available evidence argues strongly that P68 functions directly in shutting down gene activity, and consistent with such direct action it was observed that P68’s abundance increases on loci prior to and during the shutdown process (Buszczak, 2006).
How, then, does a direct role for P68 in gene shutoff relate to the additional function in RNA export observed for this molecule? Loss-of-function p68 mutations result in bristle and ovary defects similar to those seen in sbr mutants, a known component of the RNA export machinery. p68 mutant animals accumulate RNA at transcription sites scattered at many locations around the genome. Furthermore, newly transcribed and processed hsp70 transcripts accumulate at the site of transcription for prolonged periods, causing a pronounced delay in HSP70 translation. These findings suggest that P68 functions at a very early step in RNA export, or at a novel step prior to export that is required to clear completed transcripts from their sites of transcription (Buszczak, 2006).
During transcription, various proteins that mediate transcriptional elongation, RNA processing, and RNA export coat nascent transcripts to form large ribonucleoprotein complexes (mRNPs). Work in a variety of organisms suggests that the assembly of these mRNPs is under strict quality controls. Disruption of RNA processing or association of export factors with transcripts results in RNA accumulation at gene loci and the activation of the RNA surveillance pathway. Defective transcripts are targeted for degradation by the exosome, an evolutionarily conserved multisubunit RNAse complex. The exosome associates with elongation factors and localizes to actively transcribed genes in Drosophila, suggesting that transcripts are constantly monitored for defects. Interestingly, mutations in the rrp6p exosome gene not only result in the stabilization of RNAs but also lead to their improper release from chromatin. These results suggest that the exosome tethers transcripts to chromatin and ensures that only properly processed RNAs are released for export. P68 may function by interacting with nascent transcripts in a manner that confers competence for a transcript to leave its site of synthesis and undergo transport to nuclear pores. This P68-sensitive step would act as a checkpoint for final release of a completed transcript into the nucleoplasm (Buszczak, 2006).
The idea is favored that P68's requirement for gene shutoff can be explained by its proposed role as a mediator of transcript release. There are two general mechanisms by which nascent transcripts at the site of an active gene might impede transcriptional deactivation. First, some positively acting transcriptional cofactors associate with nascent transcripts rather than remaining at promoter or enhancer regions. Transcript-bound factors would remain at very high local concentrations relative to those that dissociate into the nucleoplasm prior to rebinding. Transcripts bearing such factors would be expected to promote continued gene activity, as part of an equilibrium of positively and negatively acting mechanisms that determine the instantaneous rate of transcriptional activity. During repression, up-regulated RNA release mediated by P68 would lower the local concentration of such factors and help shift the equilibrium toward a state that favors gene deactivation (Buszczak, 2006).
Gene activity is also controlled by the state of chromatin decondensation. Decondensation is correlated with high levels of histone acetylation, and ADP-ribose modification, and these modifications must be reversed to complete gene shutoff. The presence of bulky mRNPs around a gene might simply block chromatin from reassembling into the highly compact state characteristic of inactive genes. Transcripts and their associated protein complexes might also be inhibitory to chromatin-remodeling processes due to specific protein-protein interactions. The targeting of extra P68 to genes at the onset of gene shutdown might stimulate export of the remaining transcripts that would counteract the repression process (Buszczak, 2006).
Multiple genes within a chromosome region are sometimes shut down coordinately through the process of heterochromatin formation. The ability of p68 to stimulate gene deactivation via transcript release may play a role in forming heterochromatin as well as in shutting off individual genes, because p68/Lip was previously identified as a suppressor of position effect variegation (Csink. 1994). This suggests that transcript removal may be an important part of heterochromatin formation and spreading. The RNAi pathway has been shown to facilitate heterochromatic silencing. Drosophila P68 has been found in a complex with Argonaute2, suggesting that it might directly interact with the RNAi machinery (Ishizuka, 2002). There may be a mechanistic link between the function of P68 in transcript clearance and of the RNAi pathway in promoting gene silencing (Buszczak, 2006).
Drosophila has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. This study shows that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) were identified as having vital roles in this response in whole organisms. These findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals (Zambon, 2006).
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 identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).
Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).
Fmr1 is thought to have important roles in the translation of some specific mRNAs such as futsch mRNA. Although it is unclear how Fmr1 regulates translation of such mRNAs, these findings may hold some clues. Because Fmr1 interacts with ribosomal L5/5S rRNA and L11, all of which are located at the top of the 60S ribosomal subunit, it is likely that this interaction brings Fmr1 to the 60S ribosomal subunit. Therefore, the association of Fmr1 with the 60S ribosomal subunit through direct interactions with ribosomal L5/5S rRNA and L11 may inhibit translation by preventing the assembly of initiation complexes or by giving rise to structural change of the ribosomes, which, in turn, influences a step(s) after translation initiation. Alternatively, because 5S rRNA is the only known RNA species that binds ribosomal proteins, including L5, and forms a 5S RNP before it is incorporated into the ribosomes, Fmr1 may interact with the cytoplasmic nonribosome-associated 5S RNP, which, in turn, influences the formation of the mature 60S ribosomal subunit. It is interesting to note in this context that only about half of the 5S rRNA molecules in mammalian cells are associated with the 60S ribosomal subunit and that although 5S rRNA enhances ribosomal activity, it is not absolutely essential for it (Ishizuka, 2002).
Fmr1 is present in a complex isolated from S2 cells, which also contains AGO2 and Dicer. AGO2 and Dicer are essential components of RNAi. The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).
The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).
The connection that has been established between Fmr1, components of RNAi, miRNAs, and the general translation machinery is of considerable significance because they provide intriguing clues and possible connections to the function of Fmr1 and the pathways with which it may intersect. Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. For example, both the foreign dsRNAs that trigger RNAi and the endogenous miRNA precursors that function in development are processed into small RNAs by Dicer. Members of the Argonaute gene family are also involved in both the siRNA and miRNA pathways. In C. elegans, Dicer, the dsRNA-binding protein RDE-4, and a conserved DExH-box RNA helicase (DRH-1) are in a complex with RDE-1, an AGO2 ortholog. Furthermore, the human AGO2 ortholog, eIF2C2, is in a complex, the miRNP, that contains the DEAD-box RNA helicase Gem3. Therefore, Argonaute proteins appear to be in a complex that contains an RNA helicase(s), Dicer and small guide RNAs, and function in a variety of homology-dependent mechanisms that involve base-pairing between small guide RNAs and target mRNAs. The findings that Fmr1 interacts with AGO2, Dmp68, Dicer, miRNAs, and the general translation machinery, provide a means to link RNAi enzymes to translational control pathways, and are also consistent with the fact that the RISC nuclease fractionates with ribosomes (Ishizuka, 2002).
It appears that Fmr1 is important for translational control, possibly mediated by RNAi-related components and miRNAs. Although recent studies have identified a list of mRNAs that are potential FMRP targets, these results further suggest a model in which FMRP may not directly bind its mRNA targets but rather it is targeted to its mRNA substrates as part of RNAi-related apparatus, which are guided by miRNAs. How then might FMRP regulate translation of its mRNA targets? In the case of lin-4, a prototype of miRNAs, its mRNA targets (lin-14 and lin-28) are translationally repressed yet remain associated with polyribosomes, suggesting a block at a step after translation initiation. FMRP may form an miRNP complex on its mRNA targets and the association of this complex with ribosomal L5/5S rRNA and L11 may inhibit translation at one or more postinitiation steps, including elongation, termination, or the release of functional protein as discussed above. Finally, it is proposed that fragile X syndrome may be the result of protein synthesis abnormality caused by a defect(s) in an RNAi-related apparatus (Ishizuka, 2002).
This study was directed to identifying single gene mutations that are involved in trans-acting dosage regulation in order to understand further the role of such genes in aneuploid syndromes, various types of dosage compensation as well as in regulatory mechanisms. The Lighten up (Lip) gene in Drosophila melanogaster was identified in a mutagenic screen to detect dominant second site modifiers of white-blood (wbl), a retrotransposon induced allele of the white eye color locus. Lip specifically enhances the phenotype of wbl as well as a subset of other retroelement insertion alleles of white, including the copia-induced allele, white-apricot (wa), and six alleles caused by insertion of I elements. Six alleles were identified of Lip that are all recessive lethal, although phenotypically additive heteroallelic escapers were recovered in some combinations. Lip also suppresses position effect variegation, indicating that it may have a role in chromatin configuration. Additionally, Lip modifies the total transcript abundance of both the blood and copia retrotransposons, having an inverse effect on the steady state level of blood transcripts, while showing a non-additive effect on copia RNA (Csink, 1994).
The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. This study shows that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. Chromatin immunoprecipitation demonstrated that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-kappaB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity (Bates, 2005).
CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. This study shows that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function (Yao, 2010).
Search PubMed for articles about Drosophila Rm62
Bates, G. J., Nicol, S. M., Wilson, B. J., Jacobs, A. M., Bourdon, J. C., Wardrop, J., Gregory, D. J., Lane, D. P., Perkins, N. D. and Fuller-Pace, F. V. (2005). The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor. EMBO J 24: 543-553. PubMed ID: 15660129
Boeke, J., Bag, I., Ramaiah, M. J., Vetter, I., Kremmer, E., Pal-Bhadra, M., Bhadra, U. and Imhof, A. (2011). The RNA helicase Rm62 cooperates with Su(var)3-9 to re-silence active transcription in Drosophila melanogaster. PLoS One 6: e20761. PubMed ID: 21674064
Buszczak, M. and Spradling, A. C. (2006). The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin. Genes Dev 20: 977-989. PubMed ID: 16598038
Csink, A. K., Linsk, R. and Birchler, J. A. (1994). The Lighten up (Lip) gene of Drosophila melanogaster, a modifier of retroelement expression, position effect variegation and white locus insertion alleles. Genetics 138: 153-163. PubMed ID: 8001783
Cziko, A. M., McCann, C. T., Howlett, I. C., Barbee, S. A., Duncan, R. P., Luedemann, R., Zarnescu, D., Zinsmaier, K. E., Parker, R. R. and Ramaswami, M. (2009). Genetic modifiers of dFMR1 encode RNA granule components in Drosophila. Genetics 182: 1051-1060. PubMed ID: 19487564
Deddouche, S., Matt, N., Budd, A., Mueller, S., Kemp, C., Galiana-Arnoux, D., Dostert, C., Antoniewski, C., Hoffmann, J. A. and Imler, J. L. (2008). The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila. Nat Immunol 9: 1425-1432. PubMed ID: 18953338
Ding, S. W. and Voinnet, O. (2007). Antiviral immunity directed by small RNAs. Cell 130: 413-426. PubMed ID: 17693253
Fullam, A. and Schroder, M. (2013). DExD/H-box RNA helicases as mediators of anti-viral innate immunity and essential host factors for viral replication. Biochim Biophys Acta 1829: 854-865. PubMed ID: 23567047
Gerbasi, V. R., Preall, J. B., Golden, D. E., Powell, D. W., Cummins, T. D. and Sontheimer, E. J. (2011). Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis. Proc Natl Acad Sci U S A 108: 3204-3209. PubMed ID: 21300896
Ishizuka, A., Siomi, M. C. and Siomi, H. (2002). A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 16: 2497-2508. PubMed ID: 12368261
Lamiable, O., Rabhi, M., Peronnet, F., Locker, D. and Decoville, M. (2010). Rm62, a DEAD-box RNA helicase, complexes with DSP1 in Drosophila embryos. Genesis 48: 244-253. PubMed ID: 20196121
Lei, E. P. and Corces, V. G. (2006). RNA interference machinery influences the nuclear organization of a chromatin insulator. Nat. Genet. 38(8): 936-41. Medline abstract: 16862159
Loo, Y. M. and Gale, M., Jr. (2011). Immune signaling by RIG-I-like receptors. Immunity 34: 680-692. PubMed ID: 21616437
Moy, R. H., Cole, B. S., Yasunaga, A., Gold, B., Shankarling, G., Varble, A., Molleston, J. M., tenOever, B. R., Lynch, K. W. and Cherry, S. (2014). Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense. Cell 158: 764-777. PubMed ID: 25126784
Qurashi, A., Li, W., Zhou, J. Y., Peng, J. and Jin, P. (2011). Nuclear accumulation of stress response mRNAs contributes to the neurodegeneration caused by Fragile X premutation rCGG repeats. PLoS Genet 7: e1002102. PubMed ID: 21655086
Sabin, L. R., Zheng, Q., Thekkat, P., Yang, J., Hannon, G. J., Gregory, B. D., Tudor, M. and Cherry, S. (2013). Dicer-2 processes diverse viral RNA species. PLoS One 8: e55458. PubMed ID: 23424633
Yao, H., Brick, K., Evrard, Y., Xiao, T., Camerini-Otero, R. D. and Felsenfeld, G. (2010). Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA. Genes Dev 24: 2543-2555. PubMed ID: 20966046
Zambon, R. A., Vakharia, V. N. and Wu, L. P. (2006). RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster. Cell Microbiol 8: 880-889. PubMed ID: 16611236
Zhang, Z., Yuan, B., Bao, M., Lu, N., Kim, T. and Liu, Y. J. (2011). The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol 12: 959-965. PubMed ID: 21892174
date revised: 15 November 2014
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