Yeast Trf5p is a nuclear poly(A) polymerase

Recent analyses have shown that the activity of the yeast nuclear exosome is stimulated by the Trf4p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex. Here, we report that strains lacking the Rrp6p component of the nuclear exosome accumulate polyadenylated forms of many different ribosomal RNA precursors (pre-rRNAs). This polyadenylation is reduced in strains lacking either the poly(A) polymerase Trf4p or its close homologue Trf5p. In contrast, polyadenylation is enhanced by overexpression of Trf5p. Polyadenylation is also markedly increased in strains lacking the RNA helicase Mtr4p, indicating that it is required to couple poly(A) polymerase activity to degradation. Tandem affinity purification-tagged purified Trf5p showed polyadenylation activity in vitro, which was abolished by a double point mutation in the predicted catalytic site. Trf5p co-purified with Mtr4p and Air1p, indicating that it forms a complex, designated TRAMP5, that has functions that partially overlap with the TRAMP complex.     

Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome

The nuclear exosome is involved in a large number of RNA processing and surveillance pathways. RNase III cleavage intermediates destined to be 3'-processed or degraded can be detected when the Rrp6p subunit of the nuclear exosome is absent. Here we show that these processing and degradation intermediates are polyadenylated, and that their polyadenylation is dependent on the activity of Trf4p and Trf5p, two variant poly(A) polymerases. Polyadenylation of cleavage intermediates was inhibited when Trf4p was absent, and reduced to various extents in the absence of Trf5p, suggesting that these two poly(A) polymerases play functionally distinct roles in the polyadenylation of these RNA species. Finally, in the absence of Trf4p, we observed 3'-extended forms of the U4 snRNA that are similar to those observed in the absence of Rrp6p. These results suggest that polyadenylation of RNA processing intermediates plays a functional role in RNA processing pathways and is not limited to RNA surveillance functions.     

Structure/function analysis of the Saccharomyces cerevisiae Trf4/Pol sigma DNA polymerase

The Trf4p/Pol sigma DNA polymerase (formerly Trf4p/Pol kappa) couples DNA replication to the establishment of sister chromatid cohesion. The polymerase is encoded by two redundant homologs in Saccharomyces cerevisiae, TRF4 and TRF5, that together define a fourth essential nuclear DNA polymerase in yeast and probably in all eukaryotes. Here we present a thorough genetic analysis of the founding member of this novel family of DNA polymerases, TRF4. Analyses of mutants carrying 1 of 34 "surface-targeted" alanine scanning mutations in TRF4 have identified those regions required for Pol sigma's essential function, for its role in DNA double-strand break repair, and for its association with chromosomes. The data strongly support the importance of the regions of predicted structural similarity with the Pol beta superfamily as critical for Trf4p/Pol sigma's essential and repair functions. Surprisingly, five lethal mutations lie outside all polymerase homology in a C-terminal region. The protein possesses Mg2+-dependent 3' to 5' exonuclease activity. Cell cycle analysis reveals that Trf4p/Pol sigma associates with chromosomes in G1, S, and G2 phases, but that association is abolished coincident with dissolution of cohesion at the metaphase-to-anaphase transition.     

PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif

PAPD5 is one of the seven members of the family of noncanonical poly(A) polymerases in human cells. PAPD5 was shown to polyadenylate aberrant pre-ribosomal RNAs in vivo, similar to degradation-mediating polyadenylation by the noncanonical poly(A) polymerase Trf4p in yeast. PAPD5 has been reported to be also involved in the uridylation-dependent degradation of histone mRNAs. To test whether PAPD5 indeed catalyzes adenylation as well as uridylation of RNA substrates, we analyzed the in vitro properties of recombinant PAPD5 expressed in mammalian cells as well as in bacteria. Our results show that PAPD5 catalyzes the polyadenylation of different types of RNA substrates in vitro. Interestingly, PAPD5 is active without a protein cofactor, whereas its yeast homolog Trf4p is the catalytic subunit of a bipartite poly(A) polymerase in which a separate RNA-binding subunit is needed for activity. In contrast to the yeast protein, the C terminus of PAPD5 contains a stretch of basic amino acids that is involved in binding the RNA substrate.     

Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5'-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase beta (Polbeta). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, Trf4 is able to form a Schiff base intermediate with a 5'-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polbeta, Trf4 forms stable cross-links in vitro to 5'-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Delta background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.     

A new yeast poly(A) polymerase complex involved in RNA quality control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA^Met (tRNA_i^Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA_i^Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA_i^Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.     

Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA

Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man. Trf4/5 are thought to be essential DNA polymerases. We report that Cid13 is a poly(A) polymerase. Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR). cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor. We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.     

Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size

De novo DNA synthesis on poly(dT) by a novel mouse DNA polymerase, here named "DNA replicase," was examined for the synthesis of RNA which functions as a primer in the subsequent synthesis of DNA. As has been reported previously (Yagura, T., Kozu, T., and Seno, T. (1982) J. Biochem. (Tokyo) 91, 607-618), a novel RNA polymerase activity, which is distinguished from those of classical RNA polymerases, is associated with DNA replicase. The synthesis of RNA and DNA by DNA replicase (Mr = 16 X 10(4), by glycerol gradient sedimentation analysis) was greatly stimulated by a specific stimulating factor (Mr = 13 X 10(4), by glycerol gradient sedimentation analysis) which was found to consist of two subunits (Mr = 63 X 10(3), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Nearest neighbor analysis in which transfer of 32P from alpha-labeled nucleoside triphosphates to ribo- and deoxyribonucleotides was examined, showed th at RNA of 8-10 nucleotides long was covalently linked to the 5'-end of the DNA product molecule. This RNA, named initiator RNA, had a triphosphate group at its 5' terminus and its size and synthesis were little affected by the addition of high concentrations of deoxynucleoside triphosphate, while in these conditions deoxyribonucleotides were incorporated into initiator RNA to a limited extent. The characteristics of the DNA replicase and stimulating factor that cooperate to synthesize initiator RNA for subsequent DNA synthesis on single-stranded DNA are important because these components seem to be involved in a reaction required to initiate the synthesis of discontinuous earliest DNA intermediates (Okazaki fragments) in chromosomal DNA replication of eukaryotic cells.     

A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.     

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.