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• W1967821301 abstract "Depletion of any of the essential Lsm proteins, Lsm2–5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5′ and 3′ exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5′ and 3′ degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2–8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly. Depletion of any of the essential Lsm proteins, Lsm2–5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5′ and 3′ exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5′ and 3′ degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2–8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly. external transcribed spacers internal transcribed spacer small nucleolar ribonucleoprotein small nuclear ribonucleoprotein small nucleolar RNA small nuclear RNA Sm-like protein antibody hemagglutinin wild-type mitochondrial RNA processing tandem affinity purification peroxidase-anti-peroxidase kilobase The yeast 18S, 5.8S, and 25S rRNAs are transcribed by RNA polymerase I as a single precursor, the 35S pre-rRNA, which undergoes complex post-transcriptional processing to remove the external transcribed spacers (5′-ETS and 3′-ETS)1 and internal transcribed spacers (ITS1 and ITS2) to release mature rRNAs (see Fig. 1 A). This process involves multiple endonucleolytic and exonucleolytic steps (see Fig. 1 B) and is largely carried out in the nucleolus. In Saccharomyces cerevisiae, enzymes directly involved in these reactions include the endonucleases RNase MRP and Rnt1p, the 5′ → 3′ exonuclease Rat1p, and 3′ → 5′ exonucleases, including the exosome complex, Rex1p, and Rex2p (1, 2; reviewed in Refs. 3Kressler D. Linder P. de La Cruz J. Mol. Cell. Biol. 1999; 19: 7897-7912Crossref PubMed Scopus (309) Google Scholar and 4Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (655) Google Scholar). In addition to the RNA processing enzymes, around 110 other factors are known to be required for normal pre-rRNA processing in yeast. These include several small nucleolar ribonucleoprotein (snoRNP) particles, putative RNA helicases, GTPases, and many other assembly factors (5Fatica A. Tollervey D. Curr. Opin. Cell Biol. 2002; 14: 313-318Crossref PubMed Scopus (428) Google Scholar). It is very likely that these act to promote correct folding of the pre-rRNA, assembly of the ∼80 ribosomal proteins, and assembly/disassembly of the processing complexes, with processing inhibition arising as a secondary consequence of defects in the structure of the pre-ribosomal particles (see Refs. 3Kressler D. Linder P. de La Cruz J. Mol. Cell. Biol. 1999; 19: 7897-7912Crossref PubMed Scopus (309) Google Scholar and 4Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (655) Google Scholar). All of the enzymes known to process the pre-rRNA also process other RNA species. Rnt1p, the exosome, and Rex proteins generate the 3′-ends of small nuclear and small nucleolar RNAs (snRNAs and snoRNAs), whereas Xrn1p and Rat1p produce 5′-ends of intron-encoded and polycistronic snoRNAs (2van Hoof A. Lennertz P. Parker R. EMBO J. 2000; 19: 1357-1365Crossref PubMed Scopus (146) Google Scholar, 6Chanfreau G. Abou Elela S. Ares M., Jr. Guthrie C. Genes Dev. 1997; 11: 2741-2751Crossref PubMed Scopus (98) Google Scholar, 7Abou Elela S. Ares M.J. EMBO J. 1998; 17: 3738-3746Crossref PubMed Scopus (104) Google Scholar, 8Chanfreau G. Legrain P. Jacquier A. J. Mol. Biol. 1998; 248: 975-988Crossref Scopus (141) Google Scholar, 9Chanfreau G. Rotondo G. Legrain P. Jacquier A. EMBO J. 1998; 17: 3726-3737Crossref PubMed Scopus (140) Google Scholar, 10Petfalski E. Dandekar T. Henry Y. Tollervey D. Mol. Cell. Biol. 1998; 18: 1181-1189Crossref PubMed Scopus (176) Google Scholar, 11Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (492) Google Scholar, 12Seipelt R.L. Zheng B. Asuru A. Rymond B.C. Nucleic Acids Res. 1999; 27: 587-595Crossref PubMed Scopus (66) Google Scholar, 13Kufel J. Allmang C. Chanfreau G. Petfalski E. Lafontaine D.L.J. Tollervey D. Mol. Cell. Biol. 2000; 20: 5415-5424Crossref PubMed Scopus (117) Google Scholar). Likewise, degradation of many RNAs, including cytoplasmic messenger RNAs (mRNAs) and nuclear pre-mRNAs, involves pre-rRNA processing exonucleases: the exosome, Xrn1p, and Rat1p (14Hsu C.L. Stevens A. Mol. Cell. Biol. 1993; 13: 4826-4835Crossref PubMed Scopus (325) Google Scholar, 15Muhlrad D. Decker C.J. Parker R. Genes Dev. 1994; 8: 855-866Crossref PubMed Scopus (412) Google Scholar, 16Muhlrad D. Decker C.J. Parker R. Mol. Cell. Biol. 1995; 15: 2145-2156Crossref PubMed Scopus (263) Google Scholar, 17Anderson J.S. Parker R. EMBO J. 1998; 17: 1497-1506Crossref PubMed Scopus (521) Google Scholar, 18Bousquet-Antonelli C. Presutti C. Tollervey D. Cell. 2000; 102: 765-775Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Sm-like (Lsm) proteins have been identified in all kingdoms of life and participate in numerous RNA processing and degradation pathways. The Sm and Lsm complexes are all likely to form similar structures with seven-membered rings (or six in the case of Escherichia coliHfq) with a central hole, through which the RNA may pass (19Achsel T. Brahms H. Kastner B. Bachi A. Wilm M. Lührmann R. EMBO J. 1999; 18: 5789-5802Crossref PubMed Scopus (250) Google Scholar, 20Achsel T. Stark H. Lührmann R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3685-3689Crossref PubMed Scopus (107) Google Scholar, 21Collins B.M. Harrop S.J. Kornfeld G.D. Dawes I.W. Curmi P.M. Mabbutt B.C. J. Mol. Biol. 2001; 309: 915-923Crossref PubMed Scopus (81) Google Scholar, 22Törö I. Thore S. Mayer C. Basquin J. Séraphin B. Suck D. EMBO J. 2001; 20: 2293-2303Crossref PubMed Scopus (140) Google Scholar, 23Moller T. Franch T. Hojrup P. Keene D.R. Bachinger H.P. Brennan R.G. Valentin-Hansen P. Mol. Cell. 2002; 9: 23-30Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 24Zhang A. Wassarman K.M. Ortega J. Steven A.C. Storz G. Mol. Cell. 2002; 9: 11-22Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). An Lsm2–8p complex associates with U6 snRNA and is important for U6 accumulation, U6 snRNP biogenesis, and pre-mRNA splicing (19Achsel T. Brahms H. Kastner B. Bachi A. Wilm M. Lührmann R. EMBO J. 1999; 18: 5789-5802Crossref PubMed Scopus (250) Google Scholar,25Pannone B.K. Xue D. Wolin S.L. EMBO J. 1998; 17: 7442-7453Crossref PubMed Scopus (171) Google Scholar, 26Gottschalk A. Neubauer G. Banroques J. Mann M. Lührmann R. Fabrizio P. EMBO J. 1999; 18: 4535-4548Crossref PubMed Scopus (135) Google Scholar, 27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar, 28Salgado-Garrido J. Bragado-Nilsson E. Kandels-Lewis S. Séraphin B. EMBO J. 1999; 18: 3451-3462Crossref PubMed Scopus (221) Google Scholar, 29Stevens S.W. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7226-7231Crossref PubMed Scopus (118) Google Scholar). A complex of Lsm1–7p functions in cytoplasmic mRNA degradation, promoting mRNA decapping and 5′ degradation, probably via interactions with the decapping enzymes, Dcp1p and Dcp2p, and the 5′ → 3′ exonuclease Xrn1p (30Boeck R. Lapeyre B. Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 5062-5072Crossref PubMed Scopus (131) Google Scholar, 31Bouveret E. Rigaut G. Shevchenko A. Wilm M. Séraphin B. EMBO J. 2000; 19: 1661-1671Crossref PubMed Scopus (305) Google Scholar, 32Tharun S., He, W. Mayes A.E. Lennertz P. Beggs J.D. Parker R. Nature. 2000; 404: 515-518Crossref PubMed Scopus (334) Google Scholar, 33Tharun S. Parker R. Mol. Cell. 2001; 8: 1075-1083Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The Lsm1–7p complex also protects mRNA 3′-ends from premature degradation by the exosome complex of 3′ → 5′ exonucleases (34He W. Parker R. Genetics. 2001; 158: 1445-1455PubMed Google Scholar). Yeast Lsm proteins additionally associate with precursors to RNase P RNA, suggesting a direct role in its processing (28Salgado-Garrido J. Bragado-Nilsson E. Kandels-Lewis S. Séraphin B. EMBO J. 1999; 18: 3451-3462Crossref PubMed Scopus (221) Google Scholar), and with tRNA precursors (35Kufel J. Allmang C. Verdone L. Beggs J. Tollervey D. Mol. Cel. Biol. 2002; 22: 5248-5256Crossref PubMed Scopus (58) Google Scholar) but not with the mature RNase P RNA or the related MRP RNA (28Salgado-Garrido J. Bragado-Nilsson E. Kandels-Lewis S. Séraphin B. EMBO J. 1999; 18: 3451-3462Crossref PubMed Scopus (221) Google Scholar). Depletion of the Lsm proteins does not reduce the accumulation of the mature MRP or P RNAs, or the accumulation of any small nucleolar RNA (snoRNA) tested (27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar). 2J. Kufel, C. Allmang, E. Petfalski, J. Beggs, and D. Tollervey, unpublished observations. Sm-like proteins from Bacteria and Archaea have been shown to form homomeric ring structures (21Collins B.M. Harrop S.J. Kornfeld G.D. Dawes I.W. Curmi P.M. Mabbutt B.C. J. Mol. Biol. 2001; 309: 915-923Crossref PubMed Scopus (81) Google Scholar, 23Moller T. Franch T. Hojrup P. Keene D.R. Bachinger H.P. Brennan R.G. Valentin-Hansen P. Mol. Cell. 2002; 9: 23-30Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 24Zhang A. Wassarman K.M. Ortega J. Steven A.C. Storz G. Mol. Cell. 2002; 9: 11-22Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar) indicating that their general functions are universally conserved. The E. coliproteins are known to facilitate RNA-RNA interactions (23Moller T. Franch T. Hojrup P. Keene D.R. Bachinger H.P. Brennan R.G. Valentin-Hansen P. Mol. Cell. 2002; 9: 23-30Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 24Zhang A. Wassarman K.M. Ortega J. Steven A.C. Storz G. Mol. Cell. 2002; 9: 11-22Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), whereas an Archaeal Sm-like protein associates with the RNase P RNA, and its gene is located in an operon with a ribosomal protein, suggesting a role in ribosome synthesis or function (21Collins B.M. Harrop S.J. Kornfeld G.D. Dawes I.W. Curmi P.M. Mabbutt B.C. J. Mol. Biol. 2001; 309: 915-923Crossref PubMed Scopus (81) Google Scholar). Here, we report that yeast Lsm2–8p are required for maintenance of the normal order of pre-rRNA processing steps and the stability of both the pre-rRNAs and rRNAs. Growth and handling of S. cerevisiaewere by standard techniques. The transformation procedure was as described previously (36Gietz D., St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Yeast strains used and constructed in this study are listed in Table I. StrainYJK34 was constructed by PCR strategy as described (37Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1428) Google Scholar); construction was confirmed by PCR analysis, and expression of TAP-Lsm3p was tested by Western blotting using PAP antibodies.Table IYeast strains used in this workStrainGenotypeReference/noteAEMY19MATα ade2-1 his3Δ200 leu2-3,-112 trp1Δ1 ura3-1 LSM6∷HIS3(27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY22MATα ade2-1 his3Δ200 leu2-3,-112 trp1Δ1 ura3-1 LSM7∷HIS3(27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY24MATαade2-1 his3-11,-15 leu2-3,-112 trp1Δ1 ura3-1 LSM1∷TRP1(27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY28MATαade2-1 his3-11,-15 leu2-3,-112 trp1Δ1 ura3-1 HA∷LSM1(27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY31MATαade2-1 his3-11,-15 leu2-3,-112 trp1Δ1 ura3-1 LSM3∷TRP1 [pBM125-GAL1-HA-LSM3](27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY33MATα ade2-1 his3Δ200 leu2-3,-112 trp1Δ1 ura3-1 LSM2∷HIS3[pBM125-GAL1-LSM2-HA](27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY46MATα ade2-1 his3-11,-15 leu2-3,-112 trp1Δ1 ura3-1 LSM8∷TRP1[pBM125-GAL1-HA-LSM8](27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)AEMY47MATα ade2-1 his3-11,-15 leu2-3,-112 trp1Δ1 ura3-1 LSM5∷TRP1[pBM125-GAL1-HA-LSM5](27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar)MCY4MATα ade1-101 his3-Δ1 trp1-289 ura3-52 LEU2-GAL1-LSM4(72Cooper M. Johnston L.H. Beggs J. EMBO J. 1995; 14: 2066-2075Crossref PubMed Scopus (117) Google Scholar)BMA64MATα ade2-1 his3-11,-15 leu2-3,-112 trp1Δ ura3-1F. LacrouteYJV140MATα ade2 his3 leu2 trp2 ura3(73Venema J. Tollervey D. EMBO J. 1996; 15: 5701-5714Crossref PubMed Scopus (129) Google Scholar)YJK34as YJV140 butLSM3-TAP(35Kufel J. Allmang C. Verdone L. Beggs J. Tollervey D. Mol. Cel. Biol. 2002; 22: 5248-5256Crossref PubMed Scopus (58) Google Scholar)CAZY5MATαura3-52 his3Δ200 leu2Δ1 trp1 gal2 galΔ108 HIS3-GAL10-ProtA-SYF3(44Russell C.S. Ben-Yehuda S. Dix I. Kupiec M. Beggs J.D. RNA. 2000; 6: 1565-1572Crossref PubMed Scopus (52) Google Scholar) Open table in a new tab For depletion of the essential Lsm proteins, cells were harvested at intervals following a shift from RSG medium (1% Bacto-yeast extract, 2% Bacto-peptone, 2% galactose, 2% sucrose, 2% raffinose), or YPGal medium (1% Bacto-yeast extract, 2% Bacto-peptone, 2% galactose), to YPD medium (1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose). Otherwise strains were grown in YPD medium. The lsm-Δ strains were pre-grown at 23 °C and transferred to 37 °C. RNA was extracted as described previously (38Tollervey D. Mattaj I.W. EMBO J. 1987; 6: 469-476Crossref PubMed Scopus (119) Google Scholar). Northern hybridization and primer extension were as described previously (39Tollervey D. EMBO J. 1987; 6: 4169-4175Crossref PubMed Scopus (235) Google Scholar, 40Beltrame M. Tollervey D. EMBO J. 1992; 11: 1531-1542Crossref PubMed Scopus (217) Google Scholar). Standard 1.2% agarose/formaldehyde and 6% acrylamide/urea gels were used to analyze the high and low molecular weight RNA species, respectively. For RNA hybridization and primer extension, the following oligonucleotides were used: 001 (27SA-2), 5′-CCAGTTACGAAAATTCTTG; 002 (20S-2), 5′-GCTCTTTGCTCTTGCC; 003 (27SA-3), 5′-TGTTACCTCTGGGCCC; 004 (20S), 5′-CGGTTTTAATTGTCCTA; 006 (27SB), 5′-GGCCAGCAATTTCAAGTTA; 007 (25S+40), 5′-CTCCGCTTATTGATATGC; 008 (18S+34), 5′-CATGGCTTAATCTTTGAGAC; 011 (18S+186), 5′-TCTCTTCCAAAGGGTCG; 013 (RNA2.1), 5′-AGATTAGCCGCAGTTGG; 017 (5.8S+30), 5′-GCGTTGTTCATCGATGC; 020 (ITS2–5′B), 5′-TGAGAAGGAAATGACGCT; 022 (25S/3′ETS), 5′-GAAATAAAAAACAAATCAGAC; 026 (5′ETS+911), 5′-CCAGATAACTATCTTAAAAG; 029 (18S+1785), 5′-TAATGATCCTTCCGCA; 030 (18S+668), 5′-TTGGAAATCCAGTACACG; 033 (5′ETS+278), 5′-CGCTGCTCACCAATGG; 053 (3′ETS+180), 5′-TGGTACACTCTTACACAC; 471 (SmX3), 5′-ACGCCTACACGATGGTTGACCAGGCCTTTGAGGA. Whole cells extracts were prepared as described (37Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1428) Google Scholar) using extract equivalent to 1.6 × 1010cells. Affinity purification of TAP-tagged Lsm3p protein was performed as described (37Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1428) Google Scholar) with rabbit IgG-agarose beads (Sigma). Untagged isogenic strain (YJV140) was utilized as control. Immunoprecipitation from lysates of the HA::Lsm1 and isogenic wild-type (BMA64) strains was performed using rat monoclonal anti-HA high affinity Ab (Roche Molecular Biochemicals) bound to Protein G-agarose (Roche Molecular Biochemicals). Copurified RNAs were recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Precursors and mature RNAs were identified by Northern hybridizations and primer extension analysis. Metabolic labeling of RNA was performed as described previously (41Tollervey D. Lehtonen H. Carmo-Fonseca M. Hurt E.C. EMBO J. 1991; 10: 573-583Crossref PubMed Scopus (271) Google Scholar). TheGAL::lsm3 strain was pre-grown in galactose minimal medium lacking uracil and transferred to glucose minimal medium for 8.5 h. The isogenic WT strain (YJK53) was grown directly in glucose minimal medium. Cells at 0.3A 600 nm were labeled with [3H]uracil for 1 min followed by a chase with excess unlabeled uracil for 1, 2.5, 5, 10, 20, and 60 min. Pre-rRNA processing (Fig. 1) was assessed by pulse-chase labeling with [H3]uracil in theGAL-lsm3 strain in which expression of the essential Lsm3p was under GAL control. Cells were labeled with [3H]uracil for 1 min followed by a chase with excess unlabeled uracil for the times indicated (Fig. 2). Overall incorporation into pre-rRNA and rRNA was strongly reduced in the Lsm3p-depleted strain. Labeling was performed following growth on glucose medium for 8.5 h, prior to the appearance of any clear growth defect (27, 35, and data not shown), so the reduced incorporation is unlikely to be a simple consequence of growth inhibition. Early steps in pre-rRNA processing were strongly retarded in the Lsm3p-depleted strain, leading to substantial accumulation of the 35S pre-rRNA (Fig. 2 A, lanes 7–12). This was accompanied by a delay in the synthesis of the 27S and 20S pre-rRNAs. Little 27SA2 pre-RNA was synthesized in the mutant, so the 27SB pre-rRNA was presumably generated largely by cleavage at sites A3 and B1L. Mature 18S and 25S rRNAs were synthesized with considerable retardation, but their relative ratio was not clearly altered. Synthesis of the mature 5.8S rRNA was also strongly retarded in the GAL::lsm3strain (Fig. 2 B). The aberrant 23S RNA was detected together with other aberrant RNA intermediates (marked with asterisksin Fig. 2, A and B; see Fig. 1 C for the identities of aberrant pre-rRNA species seen in Lsm-depleted strains). We conclude that the depletion of Lsm3p inhibits pre-rRNA processing. The 35S pre-rRNA was present even after 60-min chase (Fig. 2 A, lane 12) and in other experiments was shown to persist at 2-, 4-, 6-, and 12-h chase time points (data not shown). This is probably a consequence of degradation of the labeled pre-rRNA and rRNAs in the Lsm3p-depleted strain, with subsequent re-incorporation of the labeled nucleotides, which cannot be chased by exogenous uracil. As previously reported (35Kufel J. Allmang C. Verdone L. Beggs J. Tollervey D. Mol. Cel. Biol. 2002; 22: 5248-5256Crossref PubMed Scopus (58) Google Scholar), synthesis of tRNAs was also retarded in the strain depleted of Lsm3p. Pre-rRNA processing was analyzed in more detail by Northern hybridization (Figs.Figure 3, Figure 4, Figure 5). The essential Lsm proteins, Lsm2–5p and Lsm8p, were placed underGAL control (strainsGAL::lsm2,GAL::lsm3,GAL::lsm4,GAL::lsm5, andGAL::lsm8) (27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar) and depleted by transferring the strains from permissive RSG medium (0-h samples) to repressive glucose medium. The genes encoding non-essential Lsm proteins, Lsm1p and Lsm6–7p, were deleted, giving rise to temperature-sensitive strains (strains lsm1-Δ,lsm6-Δ, and lsm7-Δ) (27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar), which were grown in glucose medium at 23 °C (0-h samples) and transferred to the non-permissive temperature of 37 °C. Depletion of the essential Lsm proteins leads to depletion of the U6 snRNA and the inhibition of pre-mRNA splicing (19Achsel T. Brahms H. Kastner B. Bachi A. Wilm M. Lührmann R. EMBO J. 1999; 18: 5789-5802Crossref PubMed Scopus (250) Google Scholar, 25Pannone B.K. Xue D. Wolin S.L. EMBO J. 1998; 17: 7442-7453Crossref PubMed Scopus (171) Google Scholar, 26Gottschalk A. Neubauer G. Banroques J. Mann M. Lührmann R. Fabrizio P. EMBO J. 1999; 18: 4535-4548Crossref PubMed Scopus (135) Google Scholar, 27Mayes A.E. Verdone L. Legrain P. Beggs J.D. EMBO J. 1999; 18: 4321-4331Crossref PubMed Scopus (195) Google Scholar, 28Salgado-Garrido J. Bragado-Nilsson E. Kandels-Lewis S. Séraphin B. EMBO J. 1999; 18: 3451-3462Crossref PubMed Scopus (221) Google Scholar, 29Stevens S.W. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7226-7231Crossref PubMed Scopus (118) Google Scholar). Inhibition of rRNA processing inlsm strains can potentially arise as a result of the splicing defect. Many r protein genes contain introns, and reduced r protein synthesis leads to the inhibition of ribosome synthesis (42Rosbash M. Harris P.K. Woolford J.L.J. Teem J.L. Cell. 1981; 24: 679-686Abstract Full Text PDF PubMed Scopus (88) Google Scholar,43Bromley S. Hereford L. Rosbash M. Mol. Cell. Biol. 1982; 2: 1205-1211Crossref PubMed Scopus (25) Google Scholar). We therefore compared the Lsm-depleted strain to strains depleted of known pre-mRNA splicing factors Prp45p (data not shown) (45Albers, A., Diment, A., Muraru, M., Russell, C. S., and Beggs, J. (2002) RNA (N. Y.) 8, in pressGoogle Scholar) and Syf3p (Figs. 3 and 5) (44Russell C.S. Ben-Yehuda S. Dix I. Kupiec M. Beggs J.D. RNA. 2000; 6: 1565-1572Crossref PubMed Scopus (52) Google Scholar).Figure 4Deletion of Lsm3p leads to degradation of the mature rRNAs. The wild-type strain (WT, lanes 1–3) and GAL-lsm3 strain (lanes 4–11) were grown in permissive RSG medium (0 h) and transferred to repressive, glucose medium at 30 °C for the times indicated. Probe names are shown in parentheses. RNA species are indicated.A and B, hybridization with the 25S 5′ probe (oligonucleotide 007: position +40). C and D, hybridization with the 18S rRNA 5′ probe (oligonucleotide 008; position +34). Oligonucleotides 011, 030, and 029 are complementary to the 18S rRNA at positions further 3′ (+178, +668, and +1785, respectively). These are shown in panel D as lanes 12–14 for the GAL::lsm3 strain 30 h after transfer to glucose medium. E, hybridization with 5.8S rRNA probe (oligonucleotide 017). Comigrating bands are indicated bydashes between lanes.View Large Image Figure ViewerDownload (PPT)Figure 5Normal 5′ and 3′ processing of 5.8S rRNA requires Lsm proteins. A–F, Northern analysis of 5.8S rRNA processing in lsm mutant strains. RNA was separated on a 6% polyacrylamide gel and hybridized with oligonucleotide probes. Probe names are indicated inparentheses on the left. RNA species are shownbetween two columns. Strains carryingGAL-regulated constructs (GAL::lsm, lanes 3–17) and the BMA64 wild-type strain (WT, lanes 1–2) were grown in permissive RSG medium (0 h) and transferred to repressive, glucose medium at 30 °C for the times indicated. Strains deleted for Lsm1p (lanes 26–28), Lsm6p (lanes 20–22), and Lsm7p (lanes 23–25) and the BMA64 wild-type strain (WT, lanes 18 and 19) were pre-grown at 23 °C (0 h) and transferred to 37 °C for the times indicated. Probes are: A, oligonucleotide 033, complementary to 5′-ETS around position +278; B, oligonucleotide 002, complementary to ITS1 upstream of site A2; C, oligonucleotide 003, complementary to ITS1 upstream of site A3;D, oligonucleotide 001, complementary to ITS1 downstream of site A3; E, oligonucleotide 020 complementary to the 5.8S-ITS2 boundary; F, oligonucleotide 017, complementary to the mature 5.8S. G–I, comparison of low molecular weight pre-rRNA processing and accumulation of unspliced pre-U3 RNA (U3-int) and mature U3 in GAL-lsm3 (lanes 1–5) and GAL-syf3 strains (lanes 6–10). Strains were grown and RNA was prepared as described for Fig. 4.View Large Image Figure ViewerDownload (PPT) Clear pre-rRNA processing defects were seen in theGAL::lsm2 to lsm5 andGAL::lsm8 strains at early times of depletion (shown for GAL::lsm3 in Fig. 3, the complete set of strains are shown for low molecular weight pre-rRNAs in Fig. 5). Even on galactose medium some elevation was seen in the level of the 35S pre-rRNA in theGAL::lsm3 strain, but other processing intermediates were present at wild-type levels. Processing was clearly defective 6 h after transfer to glucose medium, before the appearance of any detectable growth defect (27, 35, and data not shown). The 35S primary transcript was further elevated, accompanied by the appearance of the aberrant 23S RNA and depletion of the 27SA2 and 20S pre-rRNAs (Fig. 3, A–C). This phenotype is characteristic of the inhibition of pre-rRNA processing at sites A0 to A2. The level of the 27SB pre-rRNA was less strongly reduced at 6 h but was clearly reduced at later time points (Fig. 3 D), as were the mature 25S and 18S rRNAs (Fig. 3 E). In addition to the loss of the normal pre-rRNA processing intermediates, there was a dramatic accumulation of aberrant pre-rRNAs in the Lsm3p and other Lsm-depleted strains. These included the 23S and 21S RNAs, which have been seen in several other pre-rRNA processing mutants, as well as many unusual intermediates, some of which are indicated in Fig. 1 C. These included fragments extending from the 5′-end of the transcript to site D (5′ETS-D), from A1 to B1, and from site D to the 3′-end of the 25S rRNA (d-B2) (Fig. 3; see also Fig. 1 C). None of these species have been observed in the wild-type or reported for previously characterized pre-rRNA processing mutants. The appearance of these RNAs indicates that processing events in the 5′-ETS, ITS1, and ITS2 do not occur in the normal order following depletion of the Lsm complex. Additionally, a set of truncated and heterogeneous pre-rRNA-derived species, which are presumed to be degradation intermediates, strongly accumulated upon depletion of each of the essential Lsm proteins (bracketed and indicated with an asterisk in Fig. 3). Degradation intermediates were readily observed with probes directed against each of the pre-rRNA spacer regions, and the total signal for these heterogeneous species was equal to or greater than the pre-rRNAs in the wild-type strain, indicating substantial degradation of all regions of the pre-rRNA (Fig. 3 and data not shown). In addition, probes against the mature 18S and 25S rRNAs detected fragments apparently derived from their breakdown (Figs.3 E and 4). Following transfer of the GAL::prp45(data not shown) or GAL::syf3 strain (Fig. 3) to glucose medium, the time course and degree of pre-mRNA splicing inhibition was comparable to the Lsm3p-depleted strain (see Fig. 5 I below) and pre-rRNA processing was inhibited, with accumulation of 35S, 23S, and 21S species and reduced levels of 27SA2 20S pre-rRNAs. However, the pre-rRNA processing defect was distinctly different from that seen on Lsm3p depletion. In particular, no accumulation of aberrant intermediates or breakdown products was observed in the GAL-syf3 strain, and levels of the 20S pre-rRNA and mature rRNAs were not strongly reduced, probably because growth is inhibited for other reasons allowing a reduced rate of ribosome synthesis to maintain normal rRNA levels at the reduced growth rate. Depletion of Lsm1–7p (but not Lsm8p) also reduces 5′ degradation of cytoplasmic mRNA (32Tharun S., He, W. Mayes A.E. Lennertz P. Beggs J.D. Parker R. Nature. 2000; 404: 515-518Crossref PubMed Scopus (334) Google Scholar). Strains carrying thexrn1-Δ or dcp1-Δ mutations, which also inhibit mRNA 5′ degradation (16Muhlrad D. Decker C.J. Parker R. Mol. Cell. Biol. 1995; 15: 2145-2156Crossref PubMed Scopus (263) Google Scholar), have been extensively studied and do not result in pre-rRNA processing defects that resemble those seen in the Lsm-depleted strains (46, 47, and data not shown). Aprp2–1/xrn1-Δ strain (18Bousquet-Antonelli C. Presutti C. Tollervey D. Cell. 2000; 102: 765-775Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), which is simultaneously inhibited for splicing and mRNA turnover, also failed to show such defects (data not shown). We conclude that the inhibition of pre-mRNA splicing and mRNA turnover does not generally lead to the pre-rRNA turnover defects seen upon Lsm protein depletion. It remains formally possible that specific defects" @default.
• W1967821301 created "2016-06-24" @default.
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• W1967821301 date "2003-01-01" @default.
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• W1967821301 title "Lsm Proteins Are Required for Normal Processing and Stability of Ribosomal RNAs" @default.
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• W1967821301 doi "https://doi.org/10.1074/jbc.m208856200" @default.
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