Monitored eCLIP: high accuracy mapping of RNA-protein interactions

International audienceCLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotiny-lated linker is ligated at the 5 end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment

Structural and functional insights into CWC27/CWC22 heterodimer linking the exon junction complex to spliceosomes

International audienceHuman CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies