The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3′ end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5′ RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3′ end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3′ end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3′ endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3′ end formation

Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription

Eukaryotic mRNA precursors are processed at their 3' ends by a coupled cleavage/polyadenylation reaction. In recent years, most of the factors involved in 3'-end processing have been identified and evidence has been presented for the coupling of mRNA 3'-end formation to capping, splicing and transcription. These links are important for the quality control of the mRNA during synthesis

A comparison of mammalian and yeast pre-mRNA 3'-end processing

Many components of the mammalian and yeast pre-mRNA 3'-end-processing machinery have recently been purified and cDNAs or genes coding for these factors have been cloned. Most of the factors consist of multiple subunits, some of which serve to bind the RNA substrate, others of which are involved in forming a complex network of protein-protein interactions. Most of the mammalian 3'-end-processing factors are similar in their amino acid sequence to the yeast factors, indicating that they have a common evolutionary history

The Schizosaccharomyces pombe pla1 gene encodes a poly(A) polymerase and can functionally replace its Saccharomyces cerevisiae homologue.

We have isolated the poly(A) polymerase (PAP) encoding gene pla1 [for poly(A) polymerase] from the fission yeast Schizosaccharomyces pombe. Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP. The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus. Furthermore, pla1 rescues a S. cerevisiae PAP1 disruption mutant. An extract from the complemented strain is active in the specific in vitro polyadenylation assay. In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay. These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeasts

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation.

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing

Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other

The FIP1 gene encodes a component of a yeast pre-mRNA polyadenylation factor that directly interacts with poly(A) polymerase

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

A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

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

Sequence similarity between the 73-kilodalton protein of mammalian CPSF and a subunit of yeast polyadenylation factor I

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