RT Journal Article
SR Electronic
T1 Wild-type Splicing Factor U2AF1 inhibits splicing associated with a recurrent U2AF1 mutant in human lung cancers and is required for cell survival
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 048553
DO 10.1101/048553
A1 Dennis Liang Fei
A1 Hayley Motowski
A1 Rakesh Chatrikhi
A1 Sameer Prasad
A1 Jovian Yu
A1 Shaojian Gao
A1 Clara Kielkopf
A1 Robert K. Bradley
A1 Harold Varmus
YR 2016
UL http://biorxiv.org/content/early/2016/04/16/048553.abstract
AB The splicing factor gene, U2AF1, is recurrently mutated in a variety of human cancers, including lung adenocarcinomas. The most frequent U2AF1 mutant, U2AF1 p.Ser34Phe (S34F), induces specific changes in splicing that we collectively refer to as “S34F-associated splicing”, but it is unclear how these splicing changes are regulated. Moreover, while a wild-type U2AF1 allele is retained in all cancers expressing a U2AF1 mutation, the functional significance of the retained wild-type allele is unknown.Our analysis of published data on human lung adenocarcinomas indicates that lung adenocarcinomas carrying a U2AF1 S34F allele exhibit a wide range of mutant to wild-type U2AF1 (S34F:WT) mRNA ratios, which can be partially attributed to copy number variation at the U2AF1 locus. S34F:WT mRNA ratios, rather than absolute levels of U2AF1 S34F or total U2AF1 mRNA, correlate positively with the magnitude of S34F-associated splicing in lung adenocarcinoma transcriptomes. To examine the effect of S34F:WT ratios on S34F-associated splicing directly, we modified a human bronchial epithelial cell line so that U2AF1 S34F is expressed at one of the two endogenous U2AF1 loci and the S34F:WT mRNA ratio approximates one. By altering the levels of mutant or wild-type U2AF1 in this engineered cell line, we show that the degree of S34F-associated splicing is proportional to the ratio of S34F:WT gene products and not to absolute levels of either the mutant or wild-type factor. Further, we show that in nearly all cases, S34F-associated splicing alterations are largely explained by the different RNA binding affinities of recombinant protein complexes containing wild-type or mutant U2AF1. Together, these observations suggest that wild-type U2AF1 is a negative regulator of S34F-associated splicing, at least in part through differential binding to 3′ splice sites. Finally, we show that the U2AF1 S34F allele does not behave like some oncogenes: the mutated gene does not induce cell transformation, and lung adenocarcinoma cell lines are not dependent on it for growth in vitro or in vivo. Wild-type U2AF1, however, is absolutely required for cell survival, regardless of whether the cells carry the U2AF1 S34F allele.We conclude that wild-type U2AF1 has two important functions in U2AF1 S34F-expressing lung cancers: it controls the magnitude of S34F-associated splicing, and it is essential for cell survival.