Abstract
We report that newly synthesized mRNA poly(A) tails are matured to precise lengths by the Pab1p-dependent poly(A) nuclease (PAN) of Saccharomyces cerevisiae. These results provide evidence for an initial phase of mRNA deadenylation that is required for poly(A) tail length control. In RNA 3'-end processing extracts lacking PAN, transcripts are polyadenylated to lengths exceeding 200 nucleotides. By contrast, in extracts containing PAN, transcripts were produced with the expected wild-type poly(A) tail lengths of 60 to 80 nucleotides. The role for PAN in poly(A) tail length control in vivo was confirmed by the finding that mRNAs are produced with longer poly(A) tails in PAN-deficient yeast strains. Interestingly, wild-type yeast strains were found to produce transcripts which varied in their maximal poly(A) tail length, and this message-specific length control was lost in PAN-deficient strains. Our data support a model whereby mRNAs are polyadenylated by the 3'-end processing machinery with a long tail, possibly of default length, and then in a PAN-dependent manner, the poly(A) tails are rapidly matured to a message-specific length. The ability to control the length of the poly(A) tail for newly expressed mRNAs has the potential to be an important posttranscriptional regulatory step in gene expression.
Highlights
The majority of eukaryotic mRNAs have at their 3Ј end a poly(A) tail
We find that the poly(A) nuclease (PAN) deadenylase matures newly synthesized poly(A) tails to defined poly(A) tail lengths of 50 to 90 nucleotides and that this initial poly(A) tail-shortening phase is necessary for message-specific poly(A) tail length control in S. cerevisiae
We hypothesized that the Pab1p requirement for poly(A) tail length control in 3Ј-end processing extracts [1, 42] may be due to the necessity of Pab1p in the activation of PAN
Summary
The majority of eukaryotic mRNAs have at their 3Ј end a poly(A) tail. The mRNA poly(A) tail is synthesized in the nucleus in a reaction that is thought to be tightly coupled to RNA polymerase II transcription [18, 40, 50]. A deadenylation-dependent pathway of mRNA turnover has been proposed for both stable and unstable mRNAs in Saccharomyces cerevisiae [20, 46, 47] This pathway in yeast is modeled to require poly(A) tail shortening to 5 to 15 nucleotides and decapping of the mRNA by the Dcp1p enzyme [7, 35]. Pab1p appears to be important for the coupling of deadenylation and decapping, and it is thought that shortening of the poly(A) tail to lengths incapable of binding Pab1p is necessary for subsequent steps in the deadenylation-dependent pathway of mRNA turnover [14]. Pab1p cofractionates with CFI [32, 42], one of the four purified yeast fractions necessary for accurate RNA 3Ј-end processing in vitro, and has been shown to physically interact with Rna15p [1], an essential subunit of CFI required for both cleavage and polyadenylation [41]
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