Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p

An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination

Polyadenylation is a co-transcriptional process that modifies mRNA 3′-ends in eukaryotes. In yeast, CF IA and CPF constitute the core 3′-end maturation complex. CF IA comprises Rna14p, Rna15p, Pcf11p and Clp1p. CF IA interacts with the C-terminal domain of RNA Pol II largest subunit via Pcf11p which links pre-mRNA 3′-end processing to transcription termination. Here, we analysed the role of Clp1p in 3′ processing. Clp1p binds ATP and interacts in CF IA with Pcf11p only. Depletion of Clp1p abolishes transcription termination. Moreover, we found that association of mutations in the ATP-binding domain and in the distant Pcf11p-binding region impair 3′-end processing. Strikingly, these mutations prevent not only Clp1p-Pcf11p interaction but also association of Pcf11p with Rna14p-Rna15p. ChIP experiments showed that Rna15p cross-linking to the 3′-end of a protein-coding gene is perturbed by these mutations whereas Pcf11p is only partially affected. Our study reveals an essential role of Clp1p in CF IA organization. We postulate that Clp1p transmits conformational changes to RNA Pol II through Pcf11p to couple transcription termination and 3′-end processing. These rearrangements likely rely on the correct orientation of ATP within Clp1p

Pti1p and Ref2p found in association with the mRNA 3′ end formation complex direct snoRNA maturation

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3′ ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3′ end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3′ end processing complex, are essential for yeast snoRNA 3′ end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3′ end formation of non-polyadenylated transcripts