SRP410432 PRJNA906503 GSE218977 The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2-mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown sequence-specificity of the mRNA 3' processing machinery. Overall design: (1). N23 MPIVA on in vitro synthesized RNA library to study sequence specificity of Compound 2, the active form of JTE-607. (2). 4sU-seq to study transcription termination in HepG2 cells treated with DMSO or JTE-607. (3). PAS-seq to study poly(A) site usage in HepG2 cells treated with DMSO or JTE-607 GSE218977 pubmed 38087090