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.     

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.     

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.     

The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.     

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.     

Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.     

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.     

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.     

Surveillance of nuclear-restricted pre-ribosomes within a subnucleolar region of Saccharomyces cerevisiae

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.     

Trypanosome MTR4 is involved in rRNA processing

The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the ‘TRAMP’ complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.     

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.