The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

Abstract

SUMMARY In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

“…EJC and SRSFs are found in the same complexes (40)(41)(42)(43). MAGOH, eIF4A3, Y14, and MLN51 form the core of the EJC that binds to sequences 20 to 24 nucleotides upstream of exon-exon boundaries (43,44).…”

“…For instance, rapid activation of the c-FOS gene by EGF mediated via CTD phosphorylation has been used as a model system for inducible gene expression (54)(55)(56). Furthermore, cotranscriptional recruitment of SRSFs, which are known to associate with the EJC (43), is integral to RNA processing (52,57). Multiple SRSFs were identified as candidate CDK12-binding partners ( Fig.…”

“…MAGOH, eIF4A3, Y14, and MLN51 form the core of the EJC that binds to sequences 20 to 24 nucleotides upstream of exon-exon boundaries (43,44). The EJC is recruited to transcripts by the intron-binding protein IBP160 (45) and deposited on the RNA via eIF4A3 prior to exon ligation during splicing (46,47).…”

“…The EJC and SRSFs interact in an RNA-independent fashion (43). Furthermore, SRSFs are found together with the EJC during cotranscriptional processing on nascent transcripts and interact with CDK12 equivalently (Fig.…”

“…Given that the EJC and SRSFs associate extensively with mRNA (43,53,59,69), we also performed RNA immunoprecipitations to determine the relative enrichment of CDK12 on c-FOS transcripts (Fig. 5B).…”