Abstract
Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.
Highlights
Prior to their export to the cytoplasm, nuclear pre-mRNAs must be capped, spliced, polyadenylated, and assembled into exportcompetent messenger ribonucleoprotein particles
Through a targeted knockdown approach, we identified PABPN1, the canonical poly(A) polymerases (PAPs), PAPa and PAPc, and the nuclear exosome components RRP6 and DIS3 as central players in this rapid decay pathway
We propose that this pathway promotes the decay of nuclear mRNAs undergoing RNA quality control (QC) and polyadenylated nuclear noncoding RNAs
Summary
Prior to their export to the cytoplasm, nuclear pre-mRNAs must be capped, spliced, polyadenylated, and assembled into exportcompetent messenger ribonucleoprotein particles (mRNPs). While the mechanisms and factors involved in nuclear RNA quality control have been extensively studied in yeast models, these pathways remain largely uncharacterized in metazoans. The yeast Trf4-Air2-Mtr polyadenylation (TRAMP) complex is a co-factor for the degradation of aberrant rRNA, tRNA, snoRNAs, snRNAs, long noncoding RNAs (lncRNAs), and mRNAs [2,4,5,6,7]. In addition to polyadenylation by TRAMP, a noncanonical poly(A) polymerase not involved in 39end formation of mRNAs, predicted RNA QC targets can be hyperadenylated by Pap, the canonical poly(A) polymerase (PAP) that polyadenylates mRNA 39-ends [9,10,11,12,13], but whether this is directly linked to decay is unclear. The mechanisms linking polyadenylation with decay remain unknown, the nuclear poly(A)-binding protein (PABP), Nab, recruits the exosome to polyadenylated RNAs, suggesting that yeast PABPs couple hyperadenylation with decay [14]
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