RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons
A l'aide d'une approche méthodologique utilisant la technique d'édition génomique CRISPR–Cas9 et permettant d'analyser les isoformes de séquences d'ARN, cette étude met en évidence le rôle, dans la croissance cellulaire normale et la suppression de tumeurs, d'exons dits "poison" qui perturbent les cadres de lecture du gène hôte et qui sont généralement hautement conservés d'une espèce à l'autre
While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR–Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of ‘poison’ cassette exons, which disrupt their host genes’ reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.
Nature Genetics 2020