EJC ipaRt
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ABSTRACT:
PROVIDER: PRJEB26421 | ENA |
REPOSITORIES: ENA
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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.
2010-10-20 | GSE24012 | GEO
Project description:To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here.
2012-10-18 | GSE41154 | GEO
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. Transcriptome sequencing (RNA-Seq) of Drosophila S2 cells to monitor the effect of EJC depletion on the cellular mRNA expression profile. Each treatment (dsRNA knockdown of MAGO (CG9401), dsRNA knockdown of eIF4AIII (CG7483)) was done in biological duplicate and each sample was sequenced separately on a quad slide on the SOLiD 3.0 platform. The reference samples were treated with a dsRNA targeted to GFP.
2010-10-19 | E-GEOD-24012 | biostudies-arrayexpress
Project description:To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here. Deep sequencing based profiling of EJC RNA footprints obtained by tandem RNA immunoprecipitation (RIPiT) of RNase I digested RNA:protein complexes.
2012-10-18 | E-GEOD-41154 | biostudies-arrayexpress
Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.
2019-01-22 | GSE106907 | GEO
Project description:Exon junction complexes (EJCs) are deposited to mRNAs during splicing and displaced from mRNAs by ribosomes in the pioneer round of translation. The understanding of EJC-bound mRNA degradation before steady-state translation has been limited to nonsense-mediated mRNA decay (NMD) due to a lack of suitable methodologies. Here, we show that RNA degradome data of Arabidopsis, rice, worm and human cells all exhibit a predominant accumulation of 5′ monophosphate (5′P) ends in the canonical EJC region. Inhibition of 5′-3′ exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the 5′P ends accumulating in the EJC region, supporting the notion that these 5′P ends are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets is dependent on an NMD factor, SMG7. Together, our finding demonstrating in vivo EJC footprinting unravels the composition of the RNA degradome, and provides a new avenue for studying NMD and other mechanisms, such as microRNAs, targeting EJC-bound mRNAs for degradation before steady-state translation.
2020-02-02 | GSE118215 | GEO
Project description:This study used Drosophila melanogaster S2 cells treated with siRNA against the EJC components mago or eIF4A3, or a non-targeting control siRNA (three samples per condition). mRNA-seq libraries were prepared from the samples and the data was used to examine 'RS-exons' - exons which reconstitute a 5' splice site when they are spliced to the upstream exon. In contrast to mammalian cells, RS-exons are not recursively spliced out from Drosophila transcripts after perturbation of the EJC.
2018-10-15 | E-MTAB-7271 | biostudies-arrayexpress
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
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
Project description:Nonsense-mediated mRNA decay (NMD) is essential for removing premature termination codon (PTC)-containing transcripts from cells. Studying the NMD pathway in model organisms can help to elucidate the NMD mechanism of humans and improves our understanding of how this biologically important process has evolved. Protozoa are among the earliest branching eukaryotes. Their NMD mechanism is poorly understood and may be primordial. We demonstrate that highly conserved Upf proteins (Upf1a, Upf2, and Upf3) are involved in the NMD pathway of the ciliate, Tetrahymena thermophila. We further show that a novel protozoa-specific nuclease, Smg6L, is responsible for destroying many NMD-targeted transcripts. The transcriptome-wide identification and characterization of NMD-targeted transcripts in vegetative Tetrahymena cells showed that many have exon–exon junctions downstream of the termination codon. However, Tetrahymena homologs of exon junction complex (EJC) core components do not form a complex and are dispensable for NMD, suggesting that NMD is EJC independent in this early branching eukaryote.
2017-07-17 | GSE90899 | GEO