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The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE59327 | GEO | 2014/08/14

SECONDARY ACCESSION(S): PRJNA255082

REPOSITORIES: GEO

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The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

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.

2014-08-14 | E-GEOD-59327 | biostudies-arrayexpress

The exon junction complex controls transposable element activity by ensuring the faithful splicing of the piwi transcript

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

2014-09-08 | E-GEOD-57710 | biostudies-arrayexpress

The exon junction complex controls transposable element activity by ensuring the faithful splicing of the piwi transcript

2014-09-08 | GSE57710 | GEO

The exon junction complex is required for definition and excision of neighboring introns in Drosophila

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.

2014-12-04 | E-GEOD-58830 | biostudies-arrayexpress

The exon junction complex is required for definition and excision of neighboring introns in Drosophila

2014-12-04 | GSE58830 | GEO

Insights into transcriptome surveillance and alternative splicing regulation by the EJC-associated splicing factor RNPS1

Project description:Alternative splicing of pre-mRNAs increases the potential for regulation and complexity of gene expression. The exon junction complex (EJC) and its associated splicing factor RNPS1 were recently shown to suppress mis-splicing resulting from the usage of cryptic and reconstituted 5’ and 3’ splice sites in the vicinity of the EJC. Here, we aimed to further investigate the mechanisms underlying splicing regulation by RNPS1. A transcriptome-wide analysis identified hundreds of splice events affected by the knockdown (KD) of RNPS1 in HeLa cells. These included alternative splice site usage as well as intron retention, exon skipping and inclusion. However, only a fraction of these RNPS1-dependent splice events was fully or partially rescued by the expression of the RNPS1 RRM. These results indicated that another domain of RNPS1 is involved in the regulation of the majority of splicing events. Deletion experiments revealed that the N-terminus and S-domain, and in particular the C-terminus of RNPS1 strongly regulate these events. Several splicing factors, including SR proteins and U1 snRNP components, were strongly reduced in the interactome of RNPS1 lacking the C terminus. We conclude that RNPS1 interacts with many splicing factors to direct the assembly of EJC-dependent and-independent splicing complexes.

2022-05-31 | PXD027251 | Pride

RNA-Seq of control and RNPS1 knockdown with or without RNPS1-RRM domain rescue in HeLa Tet-Off cells

Project description:RNPS1 is a splicing regulatory protein and a component of the ASAP/PSAP complex, which is associated with the exon junction complex and modulates alternative splicing. It was previously postulated that the isolated RRM domain of RNPS1 in complex with ASAP/PSAP is able to regulate certain alternative splicing events. We aimed to investigate in HeLa Tet-Off cells which alternative splicing events are rescued by the expression of the isolated RRM domain of RNPS1 in a RNPS1 knockdown background by using RNA-Seq analyses. The rescue construct was stably integrated into the genome using the PiggyBac transposon system. As controls, either Luciferase (Luc) siRNA was used or RNPS1 was knocked down without rescue.

2022-05-06 | E-MTAB-10768 | biostudies-arrayexpress

RNA-Seq of control and RNPS1 knockdown with or without RNPS1 wildtype or RNPS1-RRM domain rescue in Flp-In T-REx 293 cells

Project description:RNPS1 is a splicing regulatory protein and a component of the ASAP/PSAP complex, which is associated with the exon junction complex and modulates alternative splicing. It was previously postulated that the isolated RRM domain of RNPS1 in complex with ASAP/PSAP is able to regulate certain alternative splicing events. We aimed to investigate in Flp-In T-REx 293 cells which alternative splicing events are rescued by the expression of the wild type RNPS1 or the isolated RRM domain of RNPS1 in a RNPS1 knockdown background by using RNA-Seq analyses. The rescue construct was stably integrated into the genome using the PiggyBac transposon system. As controls, either Luciferase (Luc) siRNA was used or RNPS1 was knocked down without rescue.

2022-05-06 | E-MTAB-10770 | biostudies-arrayexpress

Transcriptome-wide binding sites of mutually exclusive exon junction complexes

Project description:The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. In this study we show that EJC exists in two mutually exclusive compositions characterized by peripheral proteins RNPS1 and CASC3. We identified transcriptome-wide binding sites of the two alternate EJCs via RNA:protein immunoprecipitation in tandem followed by deep sequencing (RIPiT-Seq). We show that RNPS1-EJC, which is an SR-rich mega-dalton sized RNP, is the major EJC form in the nucleus. After mRNP export to the cytoplasm and before translation, the EJC undergoes a remarkable compositional and structural remodeling into an SR- and RNPS1-devoid monomeric complex that contains CASC3. The CASC3-EJC is enriched on cytoplasmic RNAs and is the main EJC form that encounters ribosome and undergoes disassembly during translation.

2018-11-19 | GSE115977 | GEO

Magoh, Y14, eIF4AIII interactomes by TurboID proximity labelling provide no evidence for a canonical EJC in (almost) intron-less trypanosomatids

Project description:In metazoans, mRNA quality is tightly monitored from transcription to translation. A key role lies with the exon junction complex (EJC) that is placed upstream of the exon-exon junction after splicing. The EJC inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and non-sense mediated decay (NMD, UPF1/2/3). The protozoan parasite Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of the three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested as an adaptation of the EJC function to mark trans-spliced mRNAs. Here we explore the interactome of Magoh, Y14, eIF4AIII in T. brucei by TurboID proximity labelling.

2024-03-25 | PXD050946 | Pride

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