If two or more exons from the same gene are differentially enriched in RNPS1 or CASC3 footprints, these exons are almost always enriched in the same alternate EJC factor ( Figure 4A). To further reveal RNA binding patterns of the alternate EJC factors, we identified exons differentially enriched in one or the other factor. These results suggest that the two alternate EJC factors bind to two distinct pools of the same RNAs. Surprisingly, despite the mutually exclusive association of RNPS1 and CASC3 with the EJC core ( Figures 1 and 2), the two proteins are often detected on the same sites on RNA, leading to their similar apparent occupancy on individual exons as well as entire transcripts ( Figures 3B, S4A, and S4B). All analysis presented below is from two well-correlated biological replicates of formaldehyde crosslinked RIPiT-seq datasets of RNPS1- and CASC3-EJC ( Figures S3G and S3H). A strong correlation is seen between crosslinked and uncrosslinked samples ( Figures S3E and S3F). Therefore, to preserve labile interactions, we performed alternate EJC RIPiT-seq from cells cross-linked with formaldehyde before cell lysis ( Figure S3D). Unlike CASC3-EJC, RNPS1-EJC interaction was susceptible to NaCl concentration > 250 mM (data not shown). Genic read counts are highly correlated between RIPiTs, where the order of IP of EJC core and alternate factors was reversed ( Figures S3B and S3C). Strand-specific RIPiT-seq libraries from ∼35–60 nucleotide footprints yielded ∼2.5–27 million reads, of which > 80% mapped uniquely to the human genome ( Table S2). Graphical AbstractĪs expected, RIPiTs for each of the alternate EJCs specifically purified the targeted complex along with the EJC core factors and yielded abundant RNA footprints ( Figure S3A). Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. We show that the EJC originates as an SR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs).
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