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

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2022-05-31 | PXD027251 | Pride

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

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

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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

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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.

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

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