CF IA Research Articles

The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex.

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.

Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP.

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.

Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast.

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.

The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3'-end formation.

Polyadenylation of premessenger RNAs occurs posttranscriptionally in the nucleus of eukaryotic cells by cleavage of the precursor and polymerization of adenosine residues. In the yeast Saccharomyces cerevisiae, the mature poly(A) tail ranges from 60 to 70 nucleotides. 3'-end processing can be reproduced in vitro with purified factors. The cleavage reaction requires cleavage factors I and II (CF I and CF II), whereas polyadenylation involves CF I, polyadenylation factor I (PFI), and poly(A) polymerase (Pap1p). CF I has recently been separated into two factors, CF IA and CF IB. We have independently purified CF IA and found that five polypeptides cofractionate with the activity. They include Rna14p, Rna15p, Pcf11p, a new protein called Clp1p, and remarkably, the major poly(A)-binding protein Pab1p. Extracts from strains where the PAB1 gene is mutated or deleted are active for cleavage but generate transcripts bearing abnormally long poly(A) tracts. Complementation with recombinant Pab1p not only restores the length of the poly(A) tails to normal, but also triggers a poly(A) shortening activity. In addition, a monoclonal Pab1p antibody prevents the formation of poly(A) tails in extracts or in a reconstituted system. Our data support the notion that Pab1p is involved in the length control of the poly(A) tails of yeast mRNAs and define a new essential function for Pab1p in the formation of mature mRNAs.

Purification of the Saccharomyces cerevisiae Cleavage/Polyadenylation Factor I: SEPARATION INTO TWO COMPONENTS THAT ARE REQUIRED FOR BOTH CLEAVAGE AND POLYADENYLATION OF mRNA 3′ ENDS

The cleavage/polyadenylation factor I (CF I) is one of four factors required for mRNA 3' end formation in the yeast Saccharomyces cerevisiae. Here we describe the purification of CF I and its separation into two components, CF IA and CF IB. Both components are needed to reconstitute CF I activity in cleavage and poly(A) addition. CF IA consists of a complex of four polypeptides of 76, 70, 50, and 38 kDa, and CF IB is a single 73-kDa polypeptide. The 76- and 38-kDa subunits of CF IA correspond to the previously identified RNA14 and RNA15 proteins. The RNA14 protein, but not the 70- or 50-kDa proteins, coimmunoprecipitates with the RNA15 protein, indicating that RNA14 and RNA15 proteins exist in a tight complex. RNA15 is the only subunit of CF I that can be cross-linked to pre-mRNA.