TRF4 is involved in polyadenylation of snRNAs in Drosophila melanogaster

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.     

A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis. Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily.     

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.     

Requirement of fission yeast Cid14 in polyadenylation of rRNAs

Polyadenylation in eukaryotes is conventionally associated with increased nuclear export, translation, and stability of mRNAs. In contrast, recent studies suggest that the Trf4 and Trf5 proteins, members of a widespread family of noncanonical poly(A) polymerases, share an essential function in Saccharomyces cerevisiae that involves polyadenylation of nuclear RNAs as part of a pathway of exosome-mediated RNA turnover. Substrates for this pathway include aberrantly modified tRNAs and precursors of snoRNAs and rRNAs. Here we show that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe. Unlike trf4 trf5 double mutants, cells lacking Cid14 are viable, though they suffer an increased frequency of chromosome missegregation. The Cid14 protein is constitutively nucleolar and is required for normal nucleolar structure. A minor population of polyadenylated rRNAs was identified. These RNAs accumulated in an exosome mutant, and their presence was largely dependent on Cid14, in line with a role for Cid14 in rRNA degradation. Surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway. Our data suggest that additional substrates may include the mRNAs of genes involved in meiotic regulation. Polyadenylation-assisted nuclear RNA turnover is therefore likely to be a common eukaryotic mechanism affecting diverse biological processes.     

Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally,suggesting a role for polymerase epsilon in sister chromatid cohesion

The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.     

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.     

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.     

Nab3 Facilitates the Function of the TRAMP Complex in RNA Processing via Recruitment of Rrp6 Independent of Nrd1

Non-coding RNAs (ncRNAs) play critical roles in gene regulation. In eukaryotic cells, ncRNAs are processed and/or degraded by the nuclear exosome, a ribonuclease complex containing catalytic subunits Dis3 and Rrp6. The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is a critical exosome cofactor in budding yeast that stimulates the exosome to process/degrade ncRNAs and human TRAMP components have recently been identified. Importantly, mutations in exosome and exosome cofactor genes cause neurodegenerative disease. How the TRAMP complex interacts with other exosome cofactors to orchestrate regulation of the exosome is an open question. To identify novel interactions of the TRAMP exosome cofactor, we performed a high copy suppressor screen of a thermosensitive air1/2 TRAMP mutant. Here, we report that the Nab3 RNA-binding protein of the Nrd1-Nab3-Sen1 (NNS) complex is a potent suppressor of TRAMP mutants. Unlike Nab3, Nrd1 and Sen1 do not suppress TRAMP mutants and Nrd1 binding is not required for Nab3-mediated suppression of TRAMP suggesting an independent role for Nab3. Critically, Nab3 decreases ncRNA levels in TRAMP mutants, Nab3-mediated suppression of air1/2 cells requires the nuclear exosome component, Rrp6, and Nab3 directly binds Rrp6. We extend this analysis to identify a human RNA binding protein, RALY, which shares identity with Nab3 and can suppress TRAMP mutants. These results suggest that Nab3 facilitates TRAMP function by recruiting Rrp6 to ncRNAs for processing/degradation independent of Nrd1. The data raise the intriguing possibility that Nab3 and Nrd1 can function independently to recruit Rrp6 to ncRNA targets, providing combinatorial flexibility in RNA processing.     

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.