RESUMO
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.
Assuntos
Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Cromatografia de Afinidade , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a Poli(A) , Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNAAssuntos
Conformação Proteica , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Drosophila melanogaster , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.
Assuntos
Polinucleotídeo Adenililtransferase/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Substâncias Macromoleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação , Polinucleotídeo Adenililtransferase/química , Polinucleotídeo Adenililtransferase/genética , Conformação Proteica , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de Poliadenilação e Clivagem de mRNARESUMO
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.
Assuntos
Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Poli(A) , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Ligação Proteica , Precursores de RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
The 3' ends of most eukaryotic messenger RNAs are generated by endonucleolytic cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) plays a central role in both steps of the processing reaction. Here, the cloning of the 73-kilodalton subunit of CPSF is reported. Sequence analyses revealed that a yeast protein (Ysh1) was highly similar to the 73-kD polypeptide. Ysh1 constitutes a new subunit of polyadenylation factor I (PFI), which has a role in yeast pre-mRNA 3'-end formation. This finding was unexpected because in contrast to CPSF, PFI is only required for the polyadenylation reaction. These results contribute to the understanding of how 3'-end processing factors may have evolved.
Assuntos
Proteínas Fúngicas/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Peso Molecular , Poli A/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Poliadenilação e Clivagem de mRNARESUMO
We have identified an essential gene, called FIP1, encoding a 327 amino acid protein interacting with yeast poly(A) polymerase (PAP1) in the two-hybrid assay. Recombinant FIP1 protein forms a 1:1 complex with PAP1 in vitro. At 37 degrees C, a thermosensitive allele of FIP1 shows a shortening of poly(A) tails and a decrease in the steady-state level of actin transcripts. When assayed for 3'-end processing in vitro, fip1 mutant extracts exhibit normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Polyadenylation activity is restored by adding polyadenylation factor I (PF I). Antibodies directed against FIP1 specifically recognize a polypeptide in these fractions. Coimmunoprecipitation experiments reveal that RNA14, a subunit of cleavage factor I (CF I), directly interacts with FIP1, but not with PAP1. We propose a model in which PF I tethers PAP1 to CF I, thereby conferring specificity to poly(A) polymerase for pre-mRNA substrates.
Assuntos
Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Actinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes Fúngicos/genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Proteínas Associadas a Pancreatite , Poli A/análise , Ligação Proteica , Desnaturação Proteica , Processamento Pós-Transcricional do RNA , RNA Mensageiro/análise , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Most eukaryotic pre-messenger RNAs are processed at their 3' ends by endonucleolytic cleavage and polyadenylation. In yeast, this processing requires polyadenylate [poly(A)] polymerase (PAP) and other proteins that have not yet been characterized. Here, mutations in the PAP1 gene were shown to be synergistically lethal with previously identified mutations in the RNA14 and RNA15 genes, which suggests that their encoded proteins participate in 3'-end processing. Indeed, extracts from ma14 and rna15 mutants were shown to be deficient in both steps of processing. Biochemical complementation experiments and reconstitution of both activities with partially purified cleavage factor I (CF I) validated the genetic prediction.
