Project description:Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity either from an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wildtype cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon de-silencing in EJC-depleted Drosophila ovaries. We propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. Our observations provide a powerful basis to dissect the molecular events that underlie the role of the EJC in splicing. Analysis of splicing defects in 3 knockdowns (siGFP [control], siACN, siTSU) in Drosohpila OSCs. PolyA RNA (biological duplicates) and total-RNA was sequenced on a Illumina HiSeq2000 in PE50 mode.
Project description:Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity either from an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wildtype cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon de-silencing in EJC-depleted Drosophila ovaries. We propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. Our observations provide a powerful basis to dissect the molecular events that underlie the role of the EJC in splicing.
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. Small-RNA libraries from two control samples and four knockdowns in germline or somatic tissues of the Drosophila melanogaster ovary.
Project description:Long introns with short exons in vertebrate genes are thought to require spliceosome assembly across exons (exon definition), rather than introns, thereby requiring transcription of an exon to splice an upstream intron. Here, we developed CoLa-seq (co-transcriptional lariat sequencing) to investigate the timing and determinants of co-transcriptional splicing genome wide. Unexpectedly, 90% of all introns, including long introns, can splice before transcription of a downstream exon, indicating that exon definition is not obligatory for most human introns. Still, splicing timing varies dramatically across introns, and various genetic elements determine this variation. Strong U2AF2 binding to the polypyrimidine tract predicts early splicing, explaining exon definition-independent splicing. Together, our findings question the essentiality of exon definition and reveal features beyond intron and exon length that are determinative for splicing timing.
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.
Project description:Germline small RNA pathways initiate silencing of repetitive elements in animals and an interplay of nuclear small RNAs and chromatin modifications maintain this silencing, protecting the germline from spreading of transposable elements. In C. elegans germline, nuclear argonaute protein HRDE-1 initiates the transcriptional silencing pathway that is crucial for long term and heritable silencing of genes and repetitive regions. Here, we show that HRDE-1 interacts with components of the splicing machinery and the exon-junction complex. One such factor is the conserved RNA helicase EMB-4/AQR that binds introns and recruits the exon-junction proteins to newly spliced RNA. Our data shows that EMB-4/AQR is required for the transcriptional silencing pathway initiated by HRDE-1 and it functions by removing the intronic barriers to silencing thorugh its helicase function.
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs at a late stage of the splicing reaction and remains associated following export to the cytoplasm. This complex is involved in several cellular post-transcriptional processes including mRNA localization, translation and degradation. The EJC plays an additional role in the splicing of a subset of genes in Drosophila and in human cells but the underlying mechanism remains to be elucidated. Here, we have found a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding follicular cells. This function is mediated specifically through the control of the splicing of the piwi transcript. In absence of RnpS1 one of the piwi intron is retained. This intron contains a weak 5’ splice site as well as degenerate transposon fragments, reminiscent of heterochromatic introns. In addition, we identified a small A/T rich region, which alters its polypyrimidine tract (PPT) and confers the RnpS1’s dependency. Finally, we showed that the removal of this intron by RnpS1 requires the initial splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of challenging introns following its initial deposition to adjacent exon junctions. In total there are 4 different conditions. Comparisons were made between piwi mutant vs control piwi and rnps1 KD vs controls RnpS1
Project description:Signaling pathways are controlled by a vast array of post-translational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a novel key element of this pathway. The EJC binds the exon-exon junctions of mRNAs, and thus far, has been linked exclusively to post-splicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC.