PUF60 and cancer: We have experimentally characterized the impact of 36 cancer-associated RRM substitutions in PUF60 or U2AF65 on protein properties and splicing in cis and trans. Identification of mutations with trans-acting splicing defects was facilitated by sensitive 3′ss previously found by RNA-seq of cells depleted of PUF60 [14], highlighting the power of this method to identify exploitable targets of important splicing factors in the whole transcriptome.