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

SummaryThrough 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 performed transcriptome-wide mapping of both binding sites of 3′ end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, 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-64τ and CFIm 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 across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown.

Graphical AbstractFigure optionsDownload as PowerPoint slideHighlights
► Thousands of poly(A) sites are identified by the A-seq method in a human cell line
► Binding sites of pre-mRNA 3′ end processing factors are mapped by PAR-CLIP
► CstF-64 and CF Im68 exhibit the highest positional binding specificity
► CF Im 68 siRNA treatment causes a global shift toward proximal poly(A) sites