Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

摘要

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. Author Summary A gene can express multiple isoforms varying in the 3’ end, a phenomenon called alternative cleavage and polyadenylation, or APA. Previous studies have indicated that most eukaryotic genes display APA and the APA profile changes under different physiological and pathological conditions. However, how APA is regulated in the cell is unclear. Here using gene knockdown and high throughput sequencing we examine how APA is regulated by factors in the machinery responsible for cleavage and polyadenylation as well as factors that play essential roles in splicing. We identify several factors that play significant roles in APA in the last exon, including CFI-25/68, PABPN1, PABPC1, Fip1 and Pcf11. We also elucidate how cleavage and polyadenylation events are regulated in introns and near the transcription start site. We uncover a group of APA events that are highly regulated by core factors as well as in cell differentiation and development. We present an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the transcription start site, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core cleavage and polyadenylation factors.