CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.     

The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.