Abstract
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.
Highlights
All eukaryotic mRNAs except for those encoding histones have a poly (A) tail at the 3 -end which confers stability and is required for export and translation of the mRNA [1,2,3,4]
Similar results were observed in 3 -RACE assays where the loss of the RACE product of BIK/NQO1 from Star-poly (A) polymerase (PAP) knockdown was rescued by stable expression of Star-PAP but not with PAP␣ (Figure 1E-G), confirming that the 3 -end formation and expression of Star-PAP target mRNAs does not require PAP␣, and that it is exclusively controlled by Star-PAP
RNA immunoprecipitation (RIP) experiment demonstrated specific association of Star-PAP with BIK or NQO1 UTR RNA in vivo but not with non-target GAPDH, and vice versa for PAP␣ (Figure 1J). These results demonstrate that Star-PAP and PAP␣ controls distinct mRNA targets
Summary
All eukaryotic mRNAs except for those encoding histones have a poly (A) tail at the 3 -end which confers stability and is required for export and translation of the mRNA [1,2,3,4]. Polyadenylation is carried out by a 3 -end processing complex in a two-step reaction - cleavage at the 3 -UTR followed by the addition of poly (A) tail (∼250 adenosine nucleotides) to the cleaved RNA in the nucleus [1,4,5,6,7]. Canonical PAPs (PAP␣/␥ ) are responsible for the general polyadenylation of nuclear pre-mRNAs [34,35,36,37,38,39] Recent studies on another nuclear PAP, Star-PAP have shown that PAPs target select mRNAs for polyadenylation [40,41], the mechanism of target mRNA selection is still unknown
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