GSE61123
Reconstitution of CPSF active in polyadenylation: Recognition of the polyadenylation signal by WDR33
Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3'' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four, CPSF160, CPSF30, hFip1 and WDR33, are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73 and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV-crosslinked to such RNAs, as can be CPSF30. Transcriptome-wide identification of WDR33 targets by PAR-CLIP shows that WDR33 specifically binds the AAUAAA signal in vivo in contrast to any of the five other CPSF subunits tested before. Thus, our data indicate that the CPSF subunit that is mainly responsible for recognizing the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies. Overall design: Transcriptome-wide profiling of WDR33 binding sites using PAR-CLIP in HEK293 cells.