It is well established that U1 snRNP inhibits the cleavage of cryptic polyadenylation site (PAS) within introns, thereby facilitating full-length mRNA transcription for numerous genes in vertebrate cells, yet the underlying mechanism remains poorly understood. Here, by using a model PAS of wdr26 mRNA, we show that U1 snRNP predominantly interferes with the association of PAS with a core 30 processing factor CstF64, which can promote the cleavage step of mRNA 30 processing. Furthermore, we provide evidence that U1A, a component of U1 snRNP, might directly interfere with CstF64 binding on PAS through its RNA binding capacity. Consistently, U1A could potentially associate with U1-suppressed intronic PASs at the transcriptome level in human cells, showing a binding peak similar to 50 nt downstream of the cleavage site, as revealed by U1A iCLIP-seq (individual-nucleotide resolution UV crosslinking and immunoprecipitation coupled with RNA sequencing) analysis. Together, our data suggest a molecular mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 30 processing. More generally, we argue that U1 snRNP might inhibit the usage of cryptic PASs through disturbing the recruitment of core 3' processing factors. (C) 2020 Elsevier Inc. All rights reserved.