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

Abstract

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.

Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

“…For the robustness test, we used the Significance Analysis of Alternative Polyadenylation (SAAP) statistical test (FDR = 10%). SAAP compares the sample sets while controlling for sequencing depth (Li et al 2015). As above, this analysis showed a consistent pattern whereby proximal APA isoforms were overrepresented in the cytoplasm relative to the nucleus (Fig.…”

An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression

“…PABPN1 interacts physically with the nuclear exosome to degrade subsets of polyadenylated lncRNA species (Beaulieu et al 2012), and these targets include both ptRNAs (Li et al 2015) and uaRNAs (Bresson et al 2015;Li et al 2015). Despite the absence of PABPN1 in our Flag-Mtr4 co-IP/MS ( Fig.…”

“…A large majority of these RNAs contain a PAS at their 3 ′ end, implying that the canonical or very similar pre-mRNA 3 ′ processing machinery is used for their polyadenylation. However, in contrast to mRNAs (which are generally more stable, efficiently exported to the cytoplasm, and translated), uaRNAs and ptRNAs as well as many other lncRNAs are typically rapidly degraded in the nucleus (Andersson et al 2014b;Li et al 2015;Schlackow et al 2017). While neither NEXT nor TRAMP is required for ptRNA and uaRNA turnover, the nuclear exosome is.…”

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia

“…Several of the activities of individual polyadenylation complex subunits are affected by phosphorylation, methylation, calmodulin, disulfide remodeling, and ubiquitin (Jacob and Rose, 1984;Thuresson et al, 1994;Colgan et al, 1996;Mizrahi and Moore, 2000;Mouland et al, 2002;Delaney et al, 2006;Ryan, 2007;Addepalli and Hunt, 2008;Ryan and Bauer, 2008;Addepalli et al, 2010;Martin et al, 2010). In addition, alterations in the activities of individual poly(A) complex subunits are often accompanied by large-scale shifts in poly(A) site choice (Jenal et al, 2012;Martin et al, 2012;Thomas et al, 2012;Duc et al, 2013;Li et al, 2015). Hypoxia is associated with transient activation of MPKs and spikes in cytosolic calcium as well as dynamics in reactive oxygen species, along with a reduction in ATP availability and a shift to anaerobic metabolism that can alter cytosolic pH (Chang et al, 2012).…”