Canonical Poly(A) Polymerase Activity Promotes the Decay of a Wide Variety of Mammalian Nuclear RNAs

Abstract

The human nuclear poly(A)-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs). In addition, PABPN1 promotes hyperadenylation by stimulating poly(A)-polymerases (PAPα/γ), but this activity has not previously been linked to the decay of endogenous transcripts. Moreover, the mechanisms underlying target specificity have remained elusive. Here, we inactivated PAP-dependent hyperadenylation in cells by two independent mechanisms and used an RNA-seq approach to identify endogenous targets. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and promoter upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD). Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. Additional investigation showed that a genetically-encoded poly(A) tail is sufficient to drive decay, suggesting that degradation occurs independently of the canonical cleavage and polyadenylation reaction. Surprisingly, treatment with transcription inhibitors uncouples polyadenylation from decay, leading to runaway hyperadenylation of nuclear decay targets. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts.

Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP

“…In addition, we explored the roles of nuclear poly(A)-binding protein Pabpn1 by generating a Pabpn1 conditional knockout mESC line (Figure S5B) as Pabpn1 mediates degradation of various nuclear ncRNAs (Bresson et al, 2015; Li et al, 2015). 2P-seq revealed that depletion of Pabpn1 also caused an increase in detectable cleavage sites in the first intron although to a lesser extent than Exosc3 deletion, and no increase in the fourth intron (Figure 3D).…”

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.…”

“…It is noteworthy that polyadenylation can have either a stabilizing effect, as typically observed with mRNAs, or a destabilizing effect, as found with many nuclear lncRNAs subject to rapid decay (Beaulieu et al 2012;Ntini et al 2013;Bresson et al 2015).…”

Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

“…The complexity of the problem is intensified by the ongoing discovery of new splicing modalities such as detained introns (DIs). DIs occur within otherwise completely spliced and polyadenylated transcripts but, in contrast to retained intron-containing transcripts, DI-containing pre-mRNAs remain in the nucleus (Boutz et al, 2015), where they are eventually post-transcriptionally spliced or degraded (Bresson et al, 2015; Yap et al, 2012). Notably, an increase in transcripts entering the DI pathway will result in reduced levels of productive coding mRNA.…”