Genome-wide analysis of pre-mRNA 3'' end processing reveals a decisive role of human cleavage factor I in the regulation of 3'' UTR length: A-seq

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3'' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we mapped transcriptome-wide both binding sites of 3'' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64tau, CF Im25, CF Im59, and CF Im68 and 3'' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64tau and CF Im proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3'' end processing factor can modulate the length of 3'' untranslated regions transcriptome-wide, and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. Overall design: 3'' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3'' end processing factors CF Im 68 and CstF-64.

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Martin G, Gruber AR, Keller W, Zavolan M