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  • RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons.

RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons.

Nature genetics (2020-01-09)
James D Thomas, Jacob T Polaski, Qing Feng, Emma J De Neef, Emma R Hoppe, Maria V McSharry, Joseph Pangallo, Austin M Gabel, Andrea E Belleville, Jacqueline Watson, Naomi T Nkinsi, Alice H Berger, Robert K Bradley
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Human CRISPR Poison Exon Knockout Library