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Proximity labelling of FLAG-TurboID- or Myc-UltraID-tagged full-length RNPS1 or RNPS1 deletion mutants followed by proteomics analysis

ABSTRACT: RNPS1 is a component of the EJC-associated ASAP and PSAP complex. We used full-length RNPS1, RNPS1 with a C-terminal deletion and the RNPS1 RRM in a proximity labeling approach. We aimed to confirm interaction partners of RNPS1 previously identified by MS of RNPS1 IPs using the same RNPS1 deletion mutants (Project 2209 and 2236). By identifying RNPS1 interaction partners we want to uncover how RNPS1 is able to fulfill its splicing regulatory functions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryo, Kidney

SUBMITTER: Prerana Wagle

LAB HEAD: Niels H. Gehring

PROVIDER: PXD032952 | Pride | 2022-05-16

REPOSITORIES: Pride

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			Action	DRS
	Q2_ColID343_2838_01.raw	Raw
	Q2_ColID343_2838_02.raw	Raw
	Q2_ColID343_2838_03.raw	Raw
	Q2_ColID343_2838_04.raw	Raw
	Q2_ColID343_2838_05.raw	Raw

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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

The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

Project description:In metazoans, the exon junction complex (EJC) is a central component of spliced messenger ribonucleoprotein particles (mRNPs). CASC3 has been reported to be a core component of the EJC and to be crucial for assembly, the splicing regulating function of the EJC and nonsense-mediated mRNA decay (NMD). However, recent evidence suggests that CASC3 functions differently from other EJC core components. To elucidate the cellular role of CASC3, we have established human cell lines in which CASC3 was inactivated by means of CRISPR-Cas9 genome editing. We show that in these cells CASC3 is dispensable for the splicing regulatory role of the EJC. However, we find that CASC3 depletion results in the upregulation of many known and novel NMD substrates, suggesting that CASC3 is required for the efficient execution of EJC-dependent NMD. Taken together, our results challenge the model of CASC3 as an assembly factor and core component of the EJC. Our data rather show that CASC3 is involved in the degradation of NMD substrates and therefore uncover the primary molecular function of CASC3 in human cells.

2021-09-09 | PXD019161 | Pride

The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

2021-02-25 | PXD015754 | Pride

Deciphering the cellular roles and dissecting functional regions of UPF3A and UPF3B in human NMD

Project description:The paralogous proteins UPF3A and UPF3B are involved in recognizing defective mRNAs that are degraded by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, UPF3A was reported to act either an NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using overexpression, knockdowns, knockouts (KO) and rescue experiments. Overexpression or knockout of UPF3A did not result in detectable changes in global NMD activity or other specific transcriptome alterations . NMD activity was also virtually unchanged in UPF3B KO cells. In contrast, the co-depletion or co-knockout of UPF3A and UPF3B resulted in a marked NMD inhibition and a global upregulation of PTC-containing transcripts. In rescue experiments UPF3B was fully functional when either UPF2- or EJC binding was impaired . However, the deletion of both interaction sites or one interaction site and a central region of UPF3B impaired its NMD function. Altogether, our work identifies critical functional domains of UPF3B and establishes redundant roles of UPF3A and UPF3B during NMD.

2022-05-04 | PXD027120 | Pride

Nonsense-mediated mRNA decay relies on "two-factor authentication" by SMG5-SMG7

Project description:Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

2021-05-08 | PXD024747 | Pride

Mass spectrometric detection of proteins associated with lymph node metastasis in T1/2 colorectal cancer from FFPE archival specimens

Project description:Surgical resection of colorectal cancers (CRC) that have not invaded beyond the bowel wall (i.e. Stage I) can achieve 5-year patient survival rates exceeding 90%. In the majority of Stage I cases with T1 (submucosal) or T2 (not beyond the muscularis propria) depth of tumour invasion, surgery alone is curative. However, for approximately 10% of resected T1/2 CRC, even though histopathology inspection of the tumour deems it to be restricted to the bowel wall, malignant cells are identified in draining lymph nodes, signifying local metastasis. These patients are classified with Stage IIIA disease and are at greater risk than Stage I patients whose tumours show similar invasive depth, but lack lymph node involvement. To counter the risk of distant malignant dissemination, Stage IIIA patients require more extensive treatment with adjuvant chemotherapy, while Stage I patients do not. In this study we aim to get a better understanding of the underlying biological pathways linked to lymph node metastasis (LNM) using discovery based MS (DIA) and RNASeq as well as IHC and PRM to verify possible protein marker. All of this was done on archival tissue samples (FFPE).

2021-01-14 | PXD020808 | Pride

The exon junction complex is restricted to a specific subset of splice junctions

Project description:We employ multi-step affinity purification followed by high-throughput sequencing to determine the location of EJC complexes assembled on a cellular transcriptome in Drosophila S2 cells, finding 6% of the intron-containing genes were not associated with EJCs, and within genes with multiple introns, only specific exon-exon junctions assembled an EJC. RIP-Seq, 3 samples

2014-11-04 | E-GEOD-33475 | biostudies-arrayexpress

Quadruple rrm experience - Transcriptomic study of a quadruple rrm mutant which belong to a new family of AGO hook proteins

Project description:rs10-01_rrm - quadruple rrm experience - What is the role of the RRM protein family in plants? Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation. Keywords: normal vs transgenic comparison 2 dye-swap - CATMA arrays

2011-04-16 | E-GEOD-21701 | biostudies-arrayexpress

The structure and target identification of the tandem RRM of yeast Npl3 bound to RNA reveal a cooperative sequence-specific recognition and an RNA chaperone role in splicing

Project description:The conserved SR-like protein Npl3 promotes the splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in vivo to determine the consensus sequence bound to each RRM in yeast. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. In addition, our data indicate a non-specific interaction of the RS domain with RNA. These structures reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contributes to cooperative RNA-binding. Structure-guided studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic centre of the Bact spliceosomal complex. Our findings provide a mechanistic role for Npl3 during spliceosome active site formation.

2022-12-01 | E-MTAB-11736 | biostudies-arrayexpress

The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

Project description:Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). We report here that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene disc large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC. whole transcriptome RNA-seq to examine mRNAs extracted from wildtype (i.e. overexpressing lacZ) and pre-EJC-defective (i.e. overexpressing tsu RNAi) wing discs, respectively.

2016-08-19 | E-GEOD-81220 | biostudies-arrayexpress

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