| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Research Center for Materials Science, and 2 Department of Chemistry, Graduate School of Science, and 3 Institute for Advanced Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
4 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Yeast tRNA splicing endonuclease consists of four subunits: Sen54p, Sen2p, Sen34p and Sen15p (Trotta et al. 1997). The endonuclease cleaves splice sites of all the intron-containing pre-tRNAs in spite of the low content level of the enzyme and of the sequence diversity of the splice sites (Trotta et al. 1997; Lowe 2006). Sen2p and Sen34p harbor two different catalytic centers; the former is responsible for cleavage of the 5' splice site and the latter for that of the 3' splice site (Trotta et al. 1997). Accumulating pieces of evidence suggest that eukaryotic and archaeal cells utilize a highly conserved system for the removal of tRNA introns. Sen2p and Sen34p possess a homologous region essential for catalytic activities, and this region is conserved among archaebacteria and several fungi, including Schizosaccharomyces pombe (Trotta et al. 1997; Li et al. 1998; Paushkin et al. 2004). In plants, Arabidopsis thaliana has several Sen2p homologues, and existence of the tRNA splicing activity has been demonstrated biochemically in wheat germ extracts (Stange et al. 1988; Akama et al. 2000). Recently, tRNA splicing endonuclease was identified in human cells. All of the four Sen proteins are conserved between yeast and human (Paushkin et al. 2004). Since vertebrate endonucleases can cleave pre-tRNAs from S. cerevisiae, the substrate specificity and catalytic mechanism of the endonuclease seem to have been well conserved during evolution (Melton et al. 1980; Paushkin et al. 2004). tRNA ligases from Arabidopsis and rice have similar ligation activity and can substitute for the yeast counterpart (Englert & Beier 2005; Wang et al. 2006). The enzyme required for the last step of tRNA splicing, 2'-phospholtransferase (Tpt1p in S. cerevisiae), is widely conserved among a variety of organisms (Spinelli et al. 1998). Again, a human homologue of this enzyme can substitute for yeast Tpt1p in vivo (Hu et al. 2003). These facts suggested that the intracellular configuration of the splicing enzymes was also similar to each other among various types of eukaryotic cells.
Splicing endonucleases of vertebrates, including that of human, were reported to exist in the nucleus (Mattoccia et al. 1979; Paushkin et al. 2004). However, we recently found that the yeast endonuclease is localized to mitochondria (Yoshihisa et al. 2003). Both immunofluorescence microscopy and subcellular fractionation indicated that Sen2p, Sen54p and the tRNA splicing activity itself were associated with the cytosolic surface of the outer mitochondrial membrane. A sen54 mutant, the product of which did not correctly localize to mitochondria, caused defects in tRNA splicing, suggesting that the proper mitochondrial localization of Sen54p is required for its function. Temperature-sensitive (ts) sen2 mutant cells accumulated pre-tRNAs in the cytoplasm at a restrictive temperature (Yoshihisa et al. 2003). These results led us to suggest that, at least in S. cerevisiae, tRNA splicing occurs in the cytoplasm, not in the nucleus. Although this interpretation is against the commonly accepted concept that most of the splicing of nuclear encoded RNAs occurs in the nucleus, it is able to solve a long-standing paradox, that is, the tRNA splicing defects in several tRNA export mutants, including those in los1 (export carrier), rna1 (RanGAP), nup116 (nucleoporin) and so on (Sharma et al. 1996; Sarkar & Hopper 1998; Sarkar et al. 1999; Grosshans et al. 2000). In these mutants, pre-tRNAs accumulate in the nucleus under restrictive conditions. It would be difficult to explain this phenomenon if pre-tRNAs were spliced only in the nucleus. Export defects per se do not block splicing, since only mature tRNAs but not pre-tRNAs are accumulated in the nucleus under certain conditions, such as the inhibition of Leu-tRNA synthetase and depletion of Utp8p (Grosshans et al. 2000; Steiner-Mosonyi et al. 2003). In contrast, only defects of mature tRNA export in los1 cells, but not defects of splicing, were suppressed by overproduction of Cca1p, which catalyzes the addition of C, C and A nucleotides to the 3' end of tRNAs and is also proposed to function as an export carrier for tRNAs (Feng & Hopper 2002). This nuclear accumulation of pre-tRNAs in the tRNA export mutants can be readily explained from the point of view of cytoplasmic splicing, since export block causes sequestration of pre-tRNAs from the splicing endonuclease. Although the cytoplasmic splicing is an attractive model, nevertheless, direct demonstration of pre-tRNA cleavage in the cytoplasm has not been achieved.
To demonstrate the cytoplasmic splicing of pretRNAs in yeast directly, we examined whether the splice sites of pretRNAs are cleaved in the cytoplasm using a reversible ts allele of sen2. A newly isolated sen2 allele, HA-sen2-42, exhibited defects in pre-tRNA splicing at a restrictive temperature and recovered the splicing activity in several hours after a shift to a permissive temperature. Northern hybridization and fluorescence in situ hybridization (FISH) analyses demonstrated that the mutant cells accumulated pre-tRNAs in the cytoplasm at the restrictive temperature, and the accumulated pre-tRNAs were diminished when the cells were shifted to the permissive temperature. To confirm that disappearance of the pre-tRNAs did not result from their degradation but from their conversion into the mature forms, we devised a "pulse-chase/hybrid-precipitation" method to trace the fate of individual tRNA species. These experiments directly demonstrated that the cytoplasmic pre-tRNAs are indeed a bona fide intermediate for mature tRNAs.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
In our present analyses, we wanted to trace the fate of pre-tRNAs accumulated in the cytoplasm. With this aim, ts mutants, the splicing activity of which could be reversibly inactivated in vivo, are required. Although the sen2-41 cells isolated in our previous work showed clear ts defects in tRNA splicing, their tRNA splicing was apparently affected by the mutation even at the permissive temperature (Yoshihisa et al. 2003). Furthermore, the tRNA splicing defects of the mutant cells expressed at the restrictive temperature lasted several hours after the cells were transferred to 23 °C (see below). Therefore, we screened for new ts mutants of sen2 with a more pronounced reversibility in pre-tRNA accumulation.
Before screening the new sen2 alleles, we analysed the mutation point of sen2-41 and found that one nucleotide at G195 in the open reading frame (ORF) of SEN2 was deleted. Since SEN2 is essential for cell viability, the frame-shift in the 5' region of the ORF would not be tolerable. However, in this particular case, the second methionine at position 72 seems to function as a cryptic translation initiation site. Indeed, while the full-size Sen2p was detected as a 42 kDa band by immunoblotting with anti-Sen2p antibodies (Fig. 1A, lane 1), a 35 kDa band was detected in immunoblotting of the sen2-41 extract (Fig. 1A, lane 2). This size is close to 35.6 kDa, which is the size of the translation product starting from the second methionine of Sen2p. This result suggests that the N-terminal portion of Sen2p is important but not essential for its full function. We then tested by plasmid shuffling whether a series of N-terminal deletion mutants of Sen2p with an HA3 tag (three tandem repeats of hemagglutin tag) supported yeast growth. Mutants with a N-terminal deletion up to 170 residues complemented the chromosomal disruption of SEN2 at 23 °C, but not at 37 °C (Fig. 1B, sector 3, HA-Sen2p(91-377) and sector 4, HA-Sen2p(171-377)). The amino acid residues 244-377 were not able to complement 
sen2 at any temperature (Fig. 1B, sector 5). Therefore, we concluded that the ts phenotypes of sen2-41 result from a deletion of the N-terminal 71 residues. It should be noted that the N-terminal region of Sen2p is not well conserved between the yeast and other organisms. This is in good contrast to the conservation of the C-terminal catalytic region (residues 207-377) among various organisms (Trotta et al. 1997; Li et al. 1998; Paushkin et al. 2004).
|
|
Localization of the pre-tRNAs accumulated in HA-sen2-42 and HA-sen2-43 cells was analysed by FISH with probes against tRNA-ProUGG. As was the case with the parental cells, pre-tRNA-ProUGG was mainly localized in the nucleus of HA-sen2-42 and HA-sen2-43 cells at 23 °C (Fig. 3, the middle column, 23 °C). However, cytoplasmic signals of the pre-tRNA in the mutant cells increased after 4 h incubation at 37 °C, while the amount and distribution of the pre-tRNA were essentially unchanged in the wild-type cells (Fig. 3, the middle column, 37 °C). No accumulation of the pre-tRNA in the nucleus was observed in the mutant strains. Localization of the mature form of tRNA-ProUGG in these two mutant cells was similar to that in their parental strain (Fig. 3, the left column). We also tested localization of other pre-tRNAs with introns (tRNA-IleUAU, tRNA-LeuCAA and tRNA-TrpCCA) and their mature forms, and obtained essentially the same results as above (not shown). Localization of intron-less tRNAs (tRNA-GluUUC and tRNA-GlyGCC), poly(A) RNA and U14 snoRNA in these mutants was similar to that in wild-type cells (not shown), indicating that the observed effects are specific to the intron-containing pre-tRNAs. These results indicate that pre-tRNA accumulation in the cytoplasm is a common phenotype of tRNA splicing endonuclease mutants.
|
We examined whether accumulation of pre-tRNAs at the restrictive temperature is reversible in any of the sen2 mutants when the mutant cells were shifted down to the permissive temperature. The sen2 mutant cells cultured at a permissive temperature (23 °C) until the log phase were incubated for 3 h at 32 °C, 35 °C or 37 °C. To avoid too severe damage of mutant cells, we tested milder restrictive conditions in addition to the usual restrictive temperature, 37 °C. The HA-sen2-43 cells were incubated at higher temperatures for 4 h instead of 3 h since the mutant cells required longer incubation periods at the restrictive temperatures to exhibit splicing phenotypes as shown in Fig. 2. Then, the cells were shifted down to 23 °C to allow recovery from tRNA splicing defects, and were harvested after 3 h incubation at 23 °C. Pre-tRNAs were detected by staining with ethidium bromide in the sample of the sen2 mutant cells as bands that migrated between 5S rRNA and mature tRNAs on urea-PAGE (Fig. 4A, p). These bands were not detected in the sample of wild-type cells (Fig. 4A, SEN2). Among the three alleles, HA-sen2-42 showed the clearest reversibility of pre-tRNA accumulation. When HA-sen2-42 cells were incubated at 32 °C to 37 °C, large amounts of pre-tRNAs were accumulated (Fig. 4A, HA-sen2-42, lanes 2, 4 and 6). Large portions of these pre-tRNAs disappeared after 3 h incubation at 23 °C (Fig. 4A, HA-sen2-42, lanes 3, 5 and 7). In particular, the level of pre-tRNAs returned to that of wild-type cells when HA-sen2-42 cells were first incubated at 32 °C (Fig. 4A, HA-sen2-42, lane 3). On the other hand, sen2-41 cells recovered the splicing activity after the transfer to 23 °C only when the cells were preincubated at 32 °C, and the recovery was incomplete (Fig. 4A, sen2-41, lanes 2 and 3). When sen2-41 cells were incubated at 35 °C or higher, high levels of pre-tRNAs in the cells were maintained during the 3 h incubation at 23 °C (Fig. 4A, sen2-41, lanes 4 and 5). In the case of HA-sen2-43 cells, moderate amounts of pre-tRNAs were accumulated if compared with the wild-type cells irrespective of the incubation temperature, and the defects persisted even after transfer to 23 °C (Fig. 4A, HA-sen2-43). These phenotypes were also confirmed by Northern blotting (Fig. 4B, not shown). Therefore, we decided to use HA-sen2-42 for further analysis as a sen2 allele with clearly reversible defects in tRNA splicing.
|
Pre-tRNAs in the cytoplasm are converted into mature tRNAs
To trace the fate of certain pre-tRNA species accumulated in HA-sen2-42 cells, we performed pulse-chase experiments followed by isolation of a specific tRNA by hybridization (hybrid-precipitation). Briefly, cells were pulse-labeled with 3H-uracil and subsequently chased in the presence of excess cold uracil. 3H-labeled RNA samples were prepared from the cells, and specific RNA species were hybridized with a biotinylated anti-sense oligonucleotide followed by isolation of the hybrids with immobilized avidin. Wild-type and HA-sen2-42 cells pretreated at 32 °C for 2.5 h were pulse-labeled with 3H-uracil for 15 min and chased for 20 or 60 min. tRNA-ProUGG was hybrid-precipitated with an anti-sense probe against its 5'-exon and analysed by urea-PAGE. As shown in Fig. 5, even in the 15-min pulse period a considerable amount of tRNA-ProUGG was converted into the mature form in wild-type cells (Fig. 5, lane 1). The amounts of mature tRNA-ProUGG relatively increased during the chase period when compared with those of pre-tRNA-ProUGG (Fig. 5, lanes 2 and 3), indicating that the pre-tRNA-ProUGG detected at the end of the pulse period was processed to the mature form during the chase period. In contrast, most of the tRNA-ProUGG molecules remained as its precursor form during the pulse period in HA-sen2-42 cells (Fig. 5, lane 4). The pre-tRNA was slowly converted into the mature form, but more than half of the tRNA-ProUGG molecules were still in the pre-tRNA phase after the 60-min chase (Fig. 5, lane 6). These results are consistent with those from the Northern and FISH analyses described above. However, 3H incorporation into both tRNA-ProUGG-related species and the total RNA fraction considerably increased in amount even after addition of excess cold uracil (Fig. 5, not shown). Similar observations have been reported previously (Warner 1991). This may have partly resulted from slow metabolic incorporation of uracil into UTP/CTP or the existence of a large exchangeable pool of UTP/CTP in yeast cells, even though uracil auxotrophic strains (ura3) were used in these experiments. Therefore, a large part of the mature tRNA molecules detected in the "chased" samples may have been transcribed during the chase period, not during the pulse period.
|
|
Finally, we performed a pulse-chase/hybrid- precipitation experiment under similar conditions (Fig. 7). HA-sen2-42 cells pre-exposed to 32 °C for 3.0 h were pulse-labeled at the same temperature for 15 min. Subsequently, the cells were chased in the presence of cold uracil for 20 min at 32 °C to allow further incorporation of 3H-uracil into the tRNA pool and the export of pre-tRNAs to the cytoplasm. Then, the cells were incubated up to 2 h at 23 °C in the presence or absence of 5 µg/mL of thiolutin. Maturation of tRNA-ProUGG during this time course was monitored by hybrid-precipitation with the 5'-exon specific probe. Under these conditions, more than 80% of the pre-tRNA labeled at the 32 °C-pulse period was consumed in the 2 h chase period at 23 °C, and a corresponding amount of the mature form appeared (Fig. 7, lane 1 vs. lanes 2–4). Essentially the same results were obtained when we monitored maturation of pre-tRNA-LeuCAA (not shown). These and other results demonstrated that cytoplasmic pre-tRNAs are the genuine intermediates for mature tRNAs, indicating that splicing occurs in the cytoplasm in S. cerevisiae.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We screened for new ts sen2 mutants that recover their splicing activities after transfer from a restrictive temperature to a permissive temperature. All of the sen2 mutants, including the new alleles of HA-sen2-42 and HA-sen2-43, accumulated pre-tRNAs in the cytoplasm at the restrictive temperature irrespective of their mutation points. These results indicate that the cytoplasmic accumulation of pre-tRNAs is a common phenotype of tRNA endonuclease mutants, that is, a consequence of a reduction in the splicing activity. On the other hand, the reversibility of the cytoplasmic accumulation of pre-tRNAs varied among the different mutant strains, and was most prominent in HA-sen2-42 cells (Fig. 4A). The cytoplasmic pre-tRNAs accumulated sen2 mutants are susceptible to consumption at the permissive temperature, suggesting that the cytoplasmic pre-tRNAs are not dead-end products that are completely isolated from the tRNA metabolic pathways.
There are two possible pathways for consumption of the cytoplasmic pool of pre-tRNAs, conversion into mature tRNAs as vital intermediates and degradation as aberrant tRNA species. The former supports cytoplasmic splicing while nuclear splicing needs the latter to explain the disappearance of pre-tRNA from the cytoplasm. Poly(A) polymerase (Trf4p) and exosome were shown to be responsible for the degradation of unstable tRNAs in the nucleus (Kadaba et al. 2004; LaCava et al. 2005; Vanácová et al. 2005). A recent report suggested another rapid degradation system functioning independently of the nuclear exosome and Trf4p (Alexandrov et al. 2006). From the view of nuclear splicing, a cytoplasmic quality control system similar to the nuclear exosome would eliminate aberrant tRNA species, such as intron-containing pre-tRNAs, from the cytoplasm in the temperature-shift experiments with HA-sen2-42 cells. However, this was not the case. Pulse-chase/hybrid-precipitation experiments demonstrated that pre-tRNA accumulated at the restrictive temperature was converted into the mature forms when the mutant cells were shifted down to the permissive temperature.
The labeling of RNA molecules with 3H-uracil or 32P-orthophosphate is hampered by the slow incorporation of radioactivity into the RNA fraction (Warner 1991), resulting in incorporation of some radioactivity into RNA molecules in the chase period. We circumvented this problem by usage of thiolutin to halt transcription. Indeed, the pre-tRNAs accumulated in HA-sen2-42 cells in the pulse period at the restrictive temperature were converted into their mature forms in the presence of thiolutin during the chase period at the permissive temperature. In the FISH analysis, cytoplasmic pre-tRNAs accumulated in HA-sen2-42 cells during incubation at the restrictive temperature disappeared more rapidly than nuclear pre-tRNAs during the 23 °C incubation irrespective of the presence or absence of inhibitors. These results imply that cytoplasmic pre-tRNAs are susceptible to the cleavage of splice sites, and the nuclear pool of pre-tRNAs sequestered from tRNA splicing endonuclease on the mitochondrial surface must be exported to the cytoplasm to be cleaved. One strong piece of evidence against cytoplasmic splicing of pre-tRNAs was that a small but detectable amount of mature tRNAs exists in the nucleus. However, we and Hopper's group recently found that mature tRNAs are shuttling between the nucleus and the cytosol (Shaheen & Hopper 2005; Takano et al. 2005). Thus, mature tRNAs detected in the nucleus are likely pre-existing mature tRNAs that already experienced cytoplasmic processing events during their biogenesis but not newly transcribed tRNAs. There is a slight possibility that the retrograde transport system would bring cytoplasmic pre-tRNAs back to the nucleus for nuclear splicing. So far, we could not directly examine whether endogenous pre-tRNAs are re-imported to the nucleus. However, this possibility is less likely because, in our heterokaryon assay between donor cells expressing tRNA-Ser from Sch. pombe and acceptor cells without expressing the tRNA, the pre-tRNA-Ser from Sch. pombe was detected in only one of the two nuclei in a heterokaryon. The results suggest that the retrograde transport system(s) may somehow discriminates pre-tRNAs from mature tRNAs. All of these results are consistent with the cytoplasmic splicing of pre-tRNAs in S. cerevisiae.
Do the next steps of tRNA splicing occur in the cytoplasm? Although pre-tRNAs can be detected in wild-type cells, tRNA exons are scarcely detected, indicating some coupling of splice-site cleavage and exon ligation. The localization of tRNA ligase is a matter of debate. Clark and Abelson claimed that tRNA ligase, Rlg1p, is localized on the inner NE (Clark & Abelson 1987). However, Rlg1p is reported to act in the non-spliceosomal splicing of HAC1 mRNA (Sidrauski et al. 1996). Rüegsegger et al. demonstrated that HAC1 mRNA stalled on cytoplasmic polysomes is the substrate of Rlg1p (Rüegsegger et al. 2001). Schwer et al. reported that yeast tRNA ligase can be substituted by a combination of T4 RNA ligase and T4 polynucleotide kinase (Schwer et al. 2004). In their report, these proteins were not targeted to the nucleus, suggesting that the machinery for the second and third steps of tRNA splicing does not need to be localized to the nucleus. In vertebrate cells, the majority of tRNA splicing endonuclease exists in the nucleus, suggesting that pre-tRNAs are spliced in the nucleus (Mattoccia et al. 1979; Paushkin et al. 2004). Perhaps, tRNA splicing endonuclease should be associated with a certain organelle in an organism, but the localization itself can be different from species to species.
We do not know yet why each organism localizes the endonuclease in different organelles. How is the enzyme localized to the surface of the yeast mitochondria? Do yeast cells tolerate tRNA splicing endonuclease in the nucleus? Further analyses and comparison of the intracellular localization of the enzymes for tRNA splicing in other organisms are required to understand the biological significance of the intracellular movement of tRNAs during their maturation. RNA biogenesis may be achieved by more dynamic and flexible collaboration between the nucleus and the cytoplasm than we expected.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
The general techniques for yeast genetics are described in Guthrie & Fink (1991). A DNA fragment corresponding to the SEN2 ORF was amplified by mutagenic PCR with an Mn2+ buffer. The amplified fragments were subcloned into a low-copy vector pCOSC12 [CEN6-ARSH4 TRP1 CUP1p :: HA-MCS] to express N-terminally HA3-tagged fusion proteins under the regulation of the CUP1 promoter. A pool of the plasmids with mutant sen2 genes was introduced into a haploid COSC04-2 (MATa
GAL2 ura3-1 leu2-3, 112 trp1-1 his3-11,15 can1-100 sen2::LEU2/pTYSC017 [CEN6-ARSH4 URA3 SEN2]), and yeast clones that lost pTYSC017 and showed ts growth were selected on the SCD medium (0.67% w/v Yeast Nitrogen Base w/o amino acids, 0.5% w/v Vitamin Assay Casamino Acids, 2% w/v glucose) with appropriate supplements and 5'-fluoroorotic acid (5'-FOA).
Northern blotting and FISH
Small RNAs were prepared from yeast cells by the hot phenol method (Wise 1991). The obtained RNA samples were subjected to urea-PAGE (10% poly acrylamide gel with 7 M urea), transferred to Hybond N+ (Amersham Biosciences) and then hybridized at 42 °C in the presence of 4 x SSC and 0.1% SDS with appropriate anti-sense probes terminally labeled with [
-32P]-ATP. The radioactivity on the membranes was detected with Imaging Plate (Fuji Film) and Storm 860 Image Analyzer (Molecular Dynamics). FISH was performed essentially as previously described (Yoshihisa et al. 2003). After chromosomal staining with 0.3 µg/mL DAPI, fluorescent images were obtained with an IX70 microscope (Olympus) equipped with a MicroMax cooled CCD camera (Roper Scientific).
Pulse-chase and hybrid-precipitation
Yeast cells (ura3 background) were cultured in the SCD medium with 20 µg/mL uracil and appropriate nutrients to the early log phase at 23 °C and incubated at 32 °C to induce phenotypes. Typically, 10–20 OD660 cells were collected and washed with the medium depleted of uracil, and cultured in the same medium for 10 min to starve the cells of uracil. The cells were pulse-labeled with [5,6-3H] uracil (Amersham Biosciences) for 15 min and chased in the presence of 20 µg/mL cold uracil. The cells were harvested, and RNAs were prepared as described above. An RNA sample prepared from 1.8 OD660 cells was dissolved in 400 µL of 4 x SSC, 0.1% SDS and mixed with 25 pmol of an appropriate anti-sense probe conjugated with biotin. After heat treatment at 75 °C for 10 min, the samples were gradually cooled to 37 °C. A 20 µL of 50% v/v Avidin D-agarose (Vector Corporation) was added and incubated with RNA–DNA hybrids at 37 °C for 2 h. Agarose beads were collected by brief centrifugation and washed three times with 1 mL of 4 x SSC, 0.1% SDS at 37 °C, once with 1 mL of 4 x SSC at room temperature and once with 0.5 mL of ice-cold 0.5 x SSC. RNAs recovered with agarose beads were eluted by heating at 65 °C for 15 min in 20 µL of urea-PAGE sample buffer containing 7.5 M urea. The samples were subjected to urea-PAGE, transferred to Hybond N+ and analyzed by radio-imaging.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
* Correspondence: E-mail: tyoshihi{at}biochem.chem.nagoya-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Alexandrov, A., Chernyakov, I., Gu, W., Hiley, S.L., Hughes, T.R., Grayhack, E.J. & Phizicky, E.M. (2006) Rapid tRNA decay can result from lack of nonessential modifications. Mol. Cell 21, 87–98.[CrossRef][Medline]
Clark, M.W. & Abelson, J. (1987) The subnuclear localization of tRNA ligase in yeast. J. Cell Biol. 105, 1515–1526.
Culver, G.M., McCraith, S.M., Consaul, S.A., Stanford, D.R. & Phizicky, E.M. (1997) A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae. J. Biol. Chem. 272, 13203–13210.
Englert, M. & Beier, H. (2005) Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins. Nucleic Acids Res. 33, 388–399.
Feng, W. & Hopper, A.K. (2002) A Los1p-independent pathway for nuclear export of intronless tRNAs in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 99, 5412–5417.
Grosshans, H., Hurt, E. & Simos, G. (2000) An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev. 14, 830–840.
Guthrie, C. & Fink, G.R., eds. (1991) Methods Enzymol., vol. 194, Guide to Yeast Genetics and Molecular Biology. San Diego, CA: Academic Press, Inc.
Hopper, A.K. & Phizicky, E.M. (2003) tRNA transfers to the limelight. Genes Dev. 17, 162–180.
Hu, Q.-D., Lu, H., Huo, K., Ying, K., Li, J., Xie, Y., Mao, Y. & Li, Y.-Y. (2003) A human homologue of the yeast gene encoding tRNA 2'-phosphotransferase: cloning, characterization and complementation analysis. Cell. Mol. Life Sci. 60, 1725–1732.[CrossRef][Medline]
Kadaba, S., Krueger, A., Trice, T., Krecic, A.M., Hinnebusch, A.G. & Anderson, J. (2004) Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae. Genes Dev. 18, 1227–1240.
Kadowaki, T., Chen, S., Hitomi, M., Jacobs, E., Kumagai, C., Liang, S., Schneiter, R., Singleton, D., Wisniewska, J. & Tartakoff, A.M. (1994) Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J. Cell Biol. 126, 649–659.
LaCava, J., Houseley, J., Saveanu, C., Petfalski, E., Thompson, E., Jacquier, A. & Tollervey, D. (2005) RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713–724.[CrossRef][Medline]
Li, H., Trotta, C.R. & Abelson, J. (1998) Crystal structure and evolution of a transfer RNA splicing enzyme. Science 280, 279–284.
Lowe, T.M. (2006) tRNAscan-SE Analysis of the Saccharomyces cerevisiae (November 2002) genome. In: A Genomic tRNA Database. <http://lowelab.ucsc.edu/GtRNAdb/>.
Mattoccia, E., Baldi, M.I., Carrara, G., Fruscoloni, P., Benedetti, P. & Tocchini-Valentini, G.P. (1979) Separation of RNA transcription and processing activities from X. laevis germinal vesicles. Cell 18, 643–648.[CrossRef][Medline]
Melton, D.A., De Robertis, E.M. & Cortese, R. (1980) Order and intracellular location of the events involved in the maturation of a spliced tRNA. Nature 284, 143–148.[CrossRef][Medline]
Paushkin, S.V., Patel, M., Furia, B.S., Peltz, S.W. & Trotta, C.R. (2004) Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA-3' end formation. Cell 117, 311–321.[CrossRef][Medline]
Peebles, C.L., Gegenheimer, P. & Abelson, J. (1983) Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell 32, 525–536.[CrossRef][Medline]
Phizicky, E.M., Schwartz, R.C. & Abelson, J. (1986) Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J. Biol. Chem. 261, 2978–2986.
Rauhut, R., Green, P.R. & Abelson, J. (1990) Yeast tRNA-splicing endonuclease is a heterotrimeric enzyme. J. Biol. Chem. 265, 18180–18184.
Rüegsegger, U., Leber, J.H. & Walter, P. (2001) Block of HAC1 mRNA translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response. Cell 107, 103–114.[CrossRef][Medline]
Sarkar, S. & Hopper, A.K. (1998) tRNA nuclear export in Saccharomyces cerevisiae: in situ hybridization analysis. Mol. Biol. Cell 9, 3041–3055.
Sarkar, S., Azad, A.K. & Hopper, A.K. (1999) Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96, 14366–14371.
Schwer, B., Sawaya, R., Ho, C.K. & Shuman, S. (2004) Portability and fidelity of RNA-repair systems. Proc. Natl. Acad. Sci. USA 101, 2788–2793.
Shaheen, H.H. & Hopper, A.K. (2005) Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 102, 11290–11295.
Sharma, K., Fabre, E., Tekotte, H., Hurt, E.C. & Tollervey, D. (1996) Yeast nucleoporin mutants are defective in pre-tRNA splicing. Mol. Cell. Biol. 16, 294–301.
Sidrauski, C., Cox, J.S. & Walter, P. (1996) tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell 87, 405–413.[CrossRef][Medline]
Spinelli, S.L., Malik, H.S., Consaul, S.A. & Phizicky, E.M. (1998) A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli. Proc. Natl. Acad. Sci. USA 95, 14136–14141.
Stange, N., Gross, H.J. & Beier, H. (1988) Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs. EMBO J. 7, 3823–3828.[Medline]
Steiner-Mosonyi, M., Leslie, D.M., Dehghani, H., Aitchison, J.D. & Mangroo, D. (2003) Utp8p is an essential intranuclear component of the nuclear tRNA export machinery of Saccharomyces cerevisiae. J. Biol. Chem. 278, 32236–32245.
Takano, A., Endo, T. & Yoshihisa, T. (2005) tRNA actively shuttles between the nucleus and cytosol in yeast. Science 309, 140–142.
Tipper, D.J. (1973) Inhibition of yeast ribonucleic acid polymerases by thiolutin. J. Bacteriol. 116, 245–256.
Trotta, C.R., Miao, F., Arn, E.A., Stevens, S.W., Ho, C.K., Rauhut, R. & Abelson, J.N. (1997) The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases. Cell 89, 849–858.[CrossRef][Medline]
Vanácová, S., Wolf, J., Martin, G., Blank, D., Dettwiler, S., Friedlein, A., Langen, H., Keith, G. & Keller, W. (2005) A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol. 3, e189.[CrossRef][Medline]
Wang, L.K., Schwer, B., Englert, M., Beier, H. & Schuman, S. (2006) Structure–function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog. Nucleic Acids Res. 34, 517–527.
Warner, J. (1991) Labeling of RNA and phosphoproteins in Saccharomyces cerevisiae. Methods Enzymol. 194, 423–428.[Medline]
Wise, J.A. (1991) Preparation and analysis of low molecular weight RNAs and small ribonucleoproteins. Methods Enzymol. 194, 405–415.[Medline]
Yoshihisa, T., Yunoki-Esaki, K., Ohshima, C., Tanaka, N. & Endo, T. (2003) Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria. Mol. Biol. Cell 14, 3266–3279.
Received: 26 October 2006
Accepted: 29 November 2006
This article has been cited by other articles:
|
|
 |
|
  M. Zamboni, D. Scarabino, and G. P. Tocchini-Valentini Splicing of mRNA mediated by tRNA sequences in mouse cells RNA, December 1, 2009; 15(12): 2122 - 2128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B.K. Eswara, A. T. McGuire, J. B. Pierce, and D. Mangroo Utp9p Facilitates Msn5p-mediated Nuclear Reexport of Retrograded tRNAs in Saccharomyces cerevisiae Mol. Biol. Cell, December 1, 2009; 20(23): 5007 - 5025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ramirez, S. Shuman, and B. Schwer Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo RNA, September 1, 2008; 14(9): 1737 - 1745. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
