FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1984866643> ?p ?o ?g. }

• W1984866643 endingPage "8770" @default.
• W1984866643 startingPage "8759" @default.
• W1984866643 abstract "Regulated mRNA decay is essential for eukaryotic survival but the mechanisms for regulating global decay and coordinating it with growth, nutrient, and environmental cues are not known. Here we show that a signal transduction pathway containing the Pkh1/Pkh2 protein kinases and one of their effector kinases, Pkc1, is required for and regulates global mRNA decay at the deadenylation step in Saccharomyces cerevisiae. Additionally, many stresses disrupt protein synthesis and release mRNAs from polysomes for incorporation into P-bodies for degradation or storage. We find that the Pkh1/2-Pkc1 pathway is also required for stress-induced P-body assembly. Control of mRNA decay and P-body assembly by the Pkh-Pkc1 pathway only occurs in nutrient-poor medium, suggesting a novel role for these processes in evolution. Our identification of a signaling pathway for regulating global mRNA decay and P-body assembly provides a means to coordinate mRNA decay with other cellular processes essential for growth and long-term survival. Mammals may use similar regulatory mechanisms because components of the decay apparatus and signaling pathways are conserved. Regulated mRNA decay is essential for eukaryotic survival but the mechanisms for regulating global decay and coordinating it with growth, nutrient, and environmental cues are not known. Here we show that a signal transduction pathway containing the Pkh1/Pkh2 protein kinases and one of their effector kinases, Pkc1, is required for and regulates global mRNA decay at the deadenylation step in Saccharomyces cerevisiae. Additionally, many stresses disrupt protein synthesis and release mRNAs from polysomes for incorporation into P-bodies for degradation or storage. We find that the Pkh1/2-Pkc1 pathway is also required for stress-induced P-body assembly. Control of mRNA decay and P-body assembly by the Pkh-Pkc1 pathway only occurs in nutrient-poor medium, suggesting a novel role for these processes in evolution. Our identification of a signaling pathway for regulating global mRNA decay and P-body assembly provides a means to coordinate mRNA decay with other cellular processes essential for growth and long-term survival. Mammals may use similar regulatory mechanisms because components of the decay apparatus and signaling pathways are conserved. Messenger RNA decay is vital for control of eukaryotic gene expression and large-scale analyses suggest that as many as half of all changes in mRNA levels during stress are due to mRNA decay (1Fan J. Yang X. Wang W. Wood 3rd, W.H. Becker K.G. Gorospe M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10611-10616Crossref PubMed Scopus (197) Google Scholar, 2Cheadle C. Fan J. Cho-Chung Y.S. Werner T. Ray J. Do L. Gorospe M. Becker K.G. BMC Genomics. 2005; 6: 75Crossref PubMed Scopus (151) Google Scholar). Progress in understanding the mechanisms of mRNA decay is notable, yet we still do not understand how global or basal decay is regulated or how decay and processing body (P-body) 3The abbreviations used are: P-bodies, processing bodies; pkhts, cells with the pkh1ts::URA and pkh2::NAT alleles; ypkts, cells with the ypk1-1ts::HIS3 ypk2-Δ1::TRP1 alleles; SGA, synthetic genetic array; PDK1, phosphoinositide-dependent protein kinase 1. formation are coupled to stresses and nutrients. In this work we identify a signal transduction pathway that regulates global mRNA decay and P-body formation during nutrient limitation. A well studied route of mRNA decay in eukaryotes, termed deadenylation-dependent decay, begins by shortening or deadenylation of the 3′ polyadenosine (poly(A)) tail, a rate-limiting step in mRNA decay (reviewed in Refs. 3Parker R. Sheth U. Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 4Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (751) Google Scholar, 5Goldstrohm A.C. Wickens M. Nat. Rev. Mol. Cell Biol. 2008; 9: 337-344Crossref PubMed Scopus (303) Google Scholar). Deadenylation in mammals is also the initiating event in other mRNA decay pathways involving AU-rich elements, destabilizing elements in coding regions, nonsense codons, and micro-RNAs (3Parker R. Sheth U. Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 4Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (751) Google Scholar, 5Goldstrohm A.C. Wickens M. Nat. Rev. Mol. Cell Biol. 2008; 9: 337-344Crossref PubMed Scopus (303) Google Scholar, 6He F. Li X. Spatrick P. Casillo R. Dong S. Jacobson A. Mol. Cell. 2003; 12: 1439-1452Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 8Shyu A.B. Wilkinson M.F. van Hoof A. EMBO J. 2008; 27: 471-481Crossref PubMed Scopus (367) Google Scholar). Once a poly(A) tail is shortened, further degradation proceeds by removal of the 5′ N7-methylguanosine (m7G) cap from the mRNA (called “decapping”) followed by 5′-to-3′ exonuclease digestion by XrnI/KemI protein. Many mRNAs are degraded by this pathway in Saccharomyces cerevisiae, which has played key roles in elucidating the mechanisms of eukaryotic mRNA decay (9Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Alternatively, a deadenylated transcript can be degraded in the 3′-to-5′ direction by the exosome (6He F. Li X. Spatrick P. Casillo R. Dong S. Jacobson A. Mol. Cell. 2003; 12: 1439-1452Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 10Coller J. Parker R. Annu. Rev. Biochem. 2004; 73: 861-890Crossref PubMed Scopus (398) Google Scholar). Deadenylation of most mRNAs in S. cerevisiae begins with removal of ∼20 bases from the poly(A) tail by the Pan2-Pan3 deadenylase (11Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (182) Google Scholar), followed by the Ccr4-Pop2-Not (12Bai Y. Salvadore C. Chiang Y.C. Collart M.A. Liu H.Y. Denis C.L. Mol. Cell. Biol. 1999; 19: 6642-6651Crossref PubMed Scopus (127) Google Scholar) deadenylase (9Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 13Daugeron M.C. Mauxion F. Séraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Crossref PubMed Scopus (166) Google Scholar), which shortens the tail to around 10 bases at which point decapping occurs (14Denis C.L. Chen J. Prog. Nucleic Acid Res. Mol. Biol. 2003; 73: 221-250Crossref PubMed Scopus (121) Google Scholar). Mammals also use this two-step deadenylation mechanism, which is likely to be conserved in most eukaryotes (15Yamashita A. Chang T.C. Yamashita Y. Zhu W. Zhong Z. Chen C.Y. Shyu A.B. Nat. Struct. Mol. Biol. 2005; 12: 1054-1063Crossref PubMed Scopus (343) Google Scholar). Decapping begins by binding of the Lsm1–7 complex to the partially deadenylated mRNA, followed by recruitment of the Dcp1/Dcp2 decapping enzyme whose activity is stimulated by the Dhh1 and Pat1 proteins (7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 16Fillman C. Lykke-Andersen J. Curr. Opin. Cell Biol. 2005; 17: 326-331Crossref PubMed Scopus (58) Google Scholar). Decapping and 5′-to-3′ exonucleolytic degradation can occur on cytoplasmic foci termed P-bodies (10Coller J. Parker R. Annu. Rev. Biochem. 2004; 73: 861-890Crossref PubMed Scopus (398) Google Scholar, 17Sheth U. Parker R. Science. 2003; 300: 805-808Crossref PubMed Scopus (1001) Google Scholar, 18Anderson P. Kedersha N. J. Cell Biol. 2006; 172: 803-808Crossref PubMed Scopus (876) Google Scholar) or can occur independently of P-bodies (19Chu C.Y. Rana T.M. PLoS Biol. 2006; 4: e210Crossref PubMed Scopus (418) Google Scholar, 20Stoecklin G. Mayo T. Anderson P. EMBO Rep. 2006; 7: 72-77Crossref PubMed Scopus (192) Google Scholar, 21Decker C.J. Teixeira D. Parker R. J. Cell Biol. 2007; 179: 437-449Crossref PubMed Scopus (360) Google Scholar, 22Eulalio A. Behm-Ansmant I. Schweizer D. Izaurralde E. Mol. Cell. Biol. 2007; 27: 3970-3981Crossref PubMed Scopus (511) Google Scholar). Recently deadenylation, decapping, and 5′-to-3′ decay have been shown to also occur on translating mRNAs in yeast cells (23Hu W. Sweet T.J. Chamnongpol S. Baker K.E. Coller J. Nature. 2009; 461: 225-229Crossref PubMed Scopus (236) Google Scholar). To survive nutrient depletion and other stresses, eukaryotes have evolved complex coping mechanisms including changes in the rate of global mRNA decay. Mammals respond to UV-B radiation, hydrogen peroxide, heat and high osmolarity by reducing the rate of mRNA decay at the deadenylation step (24Gowrishankar G. Winzen R. Dittrich-Breiholz O. Redich N. Kracht M. Holtmann H. Biol. Chem. 2006; 387: 323-327Crossref PubMed Scopus (34) Google Scholar). S. cerevisiae cells also reduce the rate of deadenylation in response to these stresses and to glucose withdrawal, a severe nutritional stress (25Teixeira D. Sheth U. Valencia-Sanchez M.A. Brengues M. Parker R. RNA. 2005; 11: 371-382Crossref PubMed Scopus (521) Google Scholar, 26Hilgers V. Teixeira D. Parker R. RNA. 2006; 12: 1835-1845Crossref PubMed Scopus (70) Google Scholar). Additionally, many stresses inhibit translation initiation and the mRNAs released from polysomes are sequestered into P-bodies (3Parker R. Sheth U. Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 4Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (751) Google Scholar, 7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 16Fillman C. Lykke-Andersen J. Curr. Opin. Cell Biol. 2005; 17: 326-331Crossref PubMed Scopus (58) Google Scholar, 27Coller J. Parker R. Cell. 2005; 122: 875-886Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). P-bodies not only mediate mRNA decay, but studies in S. cerevisiae show that P-bodies repress translation and store mRNAs for reuse (28Brengues M. Teixeira D. Parker R. Science. 2005; 310: 486-489Crossref PubMed Scopus (586) Google Scholar). P-bodies or related structures called stress granules appear to perform similar functions in mammals (3Parker R. Sheth U. Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 4Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (751) Google Scholar, 7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 29Anderson P. Kedersha N. Trends Biochem. Sci. 2008; 33: 141-150Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). We are interested in identifying new processes controlled by the Pkh1 and Pkh2 protein kinases in S. cerevisiae because of their roles in regulating growth and survival in response to nutritional cues and environmental stresses and because they are homologs of mammalian phosphoinositide-dependent protein kinase 1 (PDK1) (30Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 31Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (117) Google Scholar). Pkh1/2 typically control cellular processes by regulating AGC-type protein kinases including Pkc1, Ypk1, and Ypk2, orthologs of mammalian serum- and glucocorticoid-inducible protein kinase 1 and Sch9, an ortholog of mammalian Akts/PKBs and S6 kinases (32Dickson R.C. Sumanasekera C. Lester R.L. Prog. Lipid Res. 2006; 45: 447-465Crossref PubMed Scopus (216) Google Scholar, 33Urban J. Soulard A. Huber A. Lippman S. Mukhopadhyay D. Deloche O. Wanke V. Anrather D. Ammerer G. Riezman H. Broach J.R. De Virgilio C. Hall M.N. Loewith R. Mol. Cell. 2007; 26: 663-674Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). Mammalian PDK1 also controls AGC-type protein kinases involved in many cellular processes (34Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (671) Google Scholar). By using synthetic genetic array (SGA) analysis (35Tong A.H. Lesage G. Bader G.D. Ding H. Xu H. Xin X. Young J. Berriz G.F. Brost R.L. Chang M. Chen Y. Cheng X. Chua G. Friesen H. Goldberg D.S. Haynes J. Humphries C. He G. Hussein S. Ke L. Krogan N. Li Z. Levinson J.N. Lu H. Ménard P. Munyana C. Parsons A.B. Ryan O. Tonikian R. Roberts T. Sdicu A.M. Shapiro J. Sheikh B. Suter B. Wong S.L. Zhang L.V. Zhu H. Burd C.G. Munro S. Sander C. Rine J. Greenblatt J. Peter M. Bretscher A. Bell G. Roth F.P. Brown G.W. Andrews B. Bussey H. Boone C. Science. 2004; 303: 808-813Crossref PubMed Scopus (1658) Google Scholar) to find new cellular processes controlled by Pkh1/2, we show that the Pkh1/2-Pkc1 pathway is required for and regulates the basal rate of mRNA decay at the deadenylation step as well as the formation of P-bodies induced by stresses. Unexpectedly, global deadenylation and mRNA decay along with P-body assembly only require Pkh1/2-Pkc1 pathway activity when cells are growing on nutrient-poor medium, establishing a connection between nutrient availability and mRNA decay. Yeast strains and plasmids used in these studies are described under supplemental Tables S1 and 2. Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or synthetic medium containing 2 or 4% glucose (SD) or 2% galactose and 1% sucrose (SG) plus 0.34% yeast nitrogen base (Difco), 1% ammonium sulfate, 30 mg/liter of adenine sulfate, tryptophan, and tyrosine, and 20 mg/liter each of histidine, leucine, lysine, methionine, and uracil. Solid medium contained 2% agar. A nutrient-enriched version of SD medium contained all 20 amino acids, adenine, uracil, inositol, and para-aminobenzoic acid (36Coller J. Methods Enzymol. 2008; 448: 267-284Crossref PubMed Scopus (25) Google Scholar). Experiments were based on published procedures (36Coller J. Methods Enzymol. 2008; 448: 267-284Crossref PubMed Scopus (25) Google Scholar, 37Passos D.O. Parker R. Methods Enzymol. 2008; 448: 409-427Crossref PubMed Scopus (14) Google Scholar, 38Nissan T. Parker R. Methods Enzymol. 2008; 448: 507-520Crossref PubMed Scopus (40) Google Scholar). To shut-off transcription of reporter genes, cells were grown to an A600 nm of 0.3–0.4 in SG medium and GAL gene expression was repressed by centrifuging cells and suspending in SD (4% glucose) medium. Where indicated in the text, transcription was also shut-off by using thiolutin (4 or 8 μg/ml). RNA was isolated as previously described (36Coller J. Methods Enzymol. 2008; 448: 267-284Crossref PubMed Scopus (25) Google Scholar). A total of 20 μg of RNA from each time point was separated by electrophoresis on a 1.4% agarose gel or 6% denaturing PAGE gel. Northern analysis was performed by using 32P-labeled 5′-end oligonucleotides (5′-AATTCCCCCCCCCCCCCCCCCCA-3′, 5′-GCCCAATGCTGGTTTAGAGACGATGATAGCATTTTCTAGCTCAGCATCAGTGATCTTAGGG-3′ and 5′-CCATACCTCTACCACCGGGGTGCTTTCTGTGCTTACCG-3′ as the probes to detect MFA2pG, GAL1-L, and CYH2 mRNAs, respectively. Blots were stripped and reprobed using a 32P-labeled 5′-end oligonucleotides (5′-GTCTAGCCGCGAGGAAGG-3′) complementary to the SCR1 RNA, which served as a loading control for each lane. For experiments involving a temperature shift, cells were grown at 25 °C, switched to 39 °C, and transcription was shut-off after 30, 60, or 120 min as indicated in the figures. Radioactive signals on Northern blots were quantified by using a PhosphorImager and mRNA half-lives were calculated from mRNA signals normalized to the SCR1 signal. The role of Pkh1/2 and Pkc1 in P-body assembly was examined in the strains listed in the figure legends. Cells grown to an A600 nm of 0.3–0.4 in SD-Leu-Ura medium at 25 °C were switched to 39 °C for various times before inducing P-body formation by glucose starvation, which involved washing cells once by centrifugation and suspending in S medium lacking glucose for 10 min followed immediately by fluorescent microscopy. To induce hypotonic stress, cells were grown in SD-Leu-Ura medium, washed with and then suspended in water for 10 min, and examined by fluorescent microscopy. To induce hypertonic stress, cells were washed with and suspended in medium containing 1 m KCl for 15 min. Microscopy was performed with a Nikon ECLIPSE E600 equipped with a PLAN APO ×100, 1.40 oil immersion objective. Room temperature samples were photographed with a SPOT RT 9.0 Monochrome-6 camera using the MetaMorph (version 6.3.0) acquisition software. Images were processed by using Adobe Photoshop (7.0). Protein synthesis was measured using a slightly modified version of a published procedure (39Ashe M.P. De Long S.K. Sachs A.B. Mol. Biol. Cell. 2000; 11: 833-848Crossref PubMed Scopus (314) Google Scholar). Cells were grown at 25 °C to an A600 nm of 0.4 in SD medium, shifted to 39 °C, and at 0, 10, 30, and 60 min, triplicate 1-ml samples were incubated for 5 min at 39 °C with 2 μl of a radioactive solution containing [35S]methionine and [35S]cysteine (EXPRESS35S35S Protein Labeling Mix, NEG0700, 1150 Ci/mmol, New England Biolabs). Protein synthesis was stopped by adding 0.5 ml of 20% cold TCA to each reaction and further sample processing was as described previously (39Ashe M.P. De Long S.K. Sachs A.B. Mol. Biol. Cell. 2000; 11: 833-848Crossref PubMed Scopus (314) Google Scholar). SGA analyses were performed as previously described (40Tong A.H. Boone C. Methods Mol. Biol. 2006; 313: 171-192PubMed Google Scholar). Thiolutin (Enzo Life Science International) was dissolved in dimethyl sulfoxide at 1 mg/ml and used at a concentration of 4 or 8 μg/ml. To use the SGA method as a way to find new processes regulated by Pkh1/2 we constructed a query strain, RCD587 (pkh1ts::URA3 pkh2::NAT), carrying a temperature-sensitive pkh1 allele and a pkh2 deletion allele, because deletion of both genes is lethal (30Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Crossing this strain to the set of 4,700 viable yeast deletion mutants (35Tong A.H. Lesage G. Bader G.D. Ding H. Xu H. Xin X. Young J. Berriz G.F. Brost R.L. Chang M. Chen Y. Cheng X. Chua G. Friesen H. Goldberg D.S. Haynes J. Humphries C. He G. Hussein S. Ke L. Krogan N. Li Z. Levinson J.N. Lu H. Ménard P. Munyana C. Parsons A.B. Ryan O. Tonikian R. Roberts T. Sdicu A.M. Shapiro J. Sheikh B. Suter B. Wong S.L. Zhang L.V. Zhu H. Burd C.G. Munro S. Sander C. Rine J. Greenblatt J. Peter M. Bretscher A. Bell G. Roth F.P. Brown G.W. Andrews B. Bussey H. Boone C. Science. 2004; 303: 808-813Crossref PubMed Scopus (1658) Google Scholar) produced 140 slow-growing haploid triple mutant strains (pkh1ts::URA3 pkh2::NAT geneX::KAN). This group contained strains mutated in genes with known roles in processes controlled by Pkh1/2 thereby validating the screen. For example, Pkh1/2 phosphorylate Pkc1, which regulates cell wall biosynthesis (31Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (117) Google Scholar) via the cell integrity pathway (41Jung U.S. Levin D.E. Mol. Microbiol. 1999; 34: 1049-1057Crossref PubMed Scopus (359) Google Scholar, 42Roberts C.J. Nelson B. Marton M.J. Stoughton R. Meyer M.R. Bennett H.A. He Y.D. Dai H. Walker W.L. Hughes T.R. Tyers M. Boone C. Friend S.H. Science. 2000; 287: 873-880Crossref PubMed Scopus (721) Google Scholar, 43Lagorce A. Hauser N.C. Labourdette D. Rodriguez C. Martin-Yken H. Arroyo J. Hoheisel J.D. François J. J. Biol. Chem. 2003; 278: 20345-20357Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 44Levin D.E. Microbiol. Mol. Biol. Rev. 2005; 69: 262-291Crossref PubMed Scopus (888) Google Scholar). Our screen identified genes including ROM2, SLT2, SMI1, and BCK1 encoding components of this pathway and FKS1, GAS1, GAS4, GAS5, LAS21, YUR1, RIM21, and OST6 that encode proteins necessary for cell wall functions. A novel and unanticipated synthetic slow growing strain carried the dhh1Δ mutation. Dhh1 is a DEXD/H-box helicase that stimulates mRNA decapping, represses translation, and functions in P-body formation (27Coller J. Parker R. Cell. 2005; 122: 875-886Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 45Coller J.M. Tucker M. Sheth U. Valencia-Sanchez M.A. Parker R. RNA. 2001; 7: 1717-1727Crossref PubMed Scopus (272) Google Scholar, 46Fischer N. Weis K. EMBO J. 2002; 21: 2788-2797Crossref PubMed Scopus (140) Google Scholar, 47Tseng-Rogenski S.S. Chong J.L. Thomas C.B. Enomoto S. Berman J. Chang T.H. Nucleic Acids Res. 2003; 31: 4995-5002Crossref PubMed Scopus (33) Google Scholar). A weaker synthetic slow growth interaction occurred with the KEM1/XRN1 gene, responsible for the 5′ to 3′ degradation of decapped mRNAs (48Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (652) Google Scholar). These data suggested that Pkh1/2 have a role in mRNA decay. To verify the DHH1 interaction, the pkh1ts pkh2Δ query strain (referred to as pkhts) was transformed with a CEN vector carrying PKH1 under control of the GAL1 promoter to make PKH1 expression galactose-inducible and glucose-repressible. DHH1 was deleted in these cells to produce pkh1ts pkh2Δ dhh1::KAN (pGAL1-PKH1) cells whose growth was more impaired at 30 °C than either parent or wild-type cells when GAL1-PKH1 expression was repressed by glucose (Fig. 1A, compare the 30 °C Glu and Gal panels), thus, validating the slow-growth phenotype found in the SGA assay. To determine whether Pkh1/2 regulates mRNA decay, we measured the half-life (t½) of the MFA2pG reporter mRNA by Northern blotting (49Decker C.J. Parker R. Genes Dev. 1993; 7: 1632-1643Crossref PubMed Scopus (527) Google Scholar). In these and subsequent experiments, transcription of the plasmid-borne GAL1-MFA2pG gene was activated by growing cells in the presence of galactose and shutoff by adding glucose to the culture medium. In pkhts mutant cells (pkh1ts pkh2Δ) grown at 25 °C the MFA2pG mRNA had a average t½ of 4.4 min, similar to the average t½ of 4.9 min in wild-type cells (Fig. 1B, 0 min time point). In pkhts cells the rate of decay became slower the longer the cells were at the restrictive temperature of 39 °C so that after 30 min of incubation the t½ was 6.7 min and after 120 min it was >30 min (Fig. 1B). In contrast, the t½ did not change in wild-type cells over this time course. The longer t½ in pkhts cells is due to reduced Pkh activity because the wild-type t½ is nearly restored when the PKH2 gene is returned to mutant cells on a single-copy plasmid (Fig. 1B). These results show that Pkh1/2 regulate MFA2pG mRNA decay. To determine whether the behavior of the MFA2pG mRNA in pkhts cells is representative of global mRNA decay, two endogenous mRNAs were examined. The GAL1 mRNA had an average t½ of 8.4 min in wild-type cells at time 0 and this value did not change during 2 h of incubation at the restrictive temperature (Fig. 1C). In pkhts cells the average t½ was 8 min at time 0 but slowed to 13.9 min following 30 min incubation at 39 °C and continued to decrease to about 26 min after 2 h. The same trends were seen with the CYH2 mRNA with the t½ remaining relatively constant in wild-type cells (average of about 8.5 min) and becoming longer in pkhts cells during the 2-h incubation at 39 °C (Fig. 1D). For both of these endogenous mRNAs the slowing of the decay rate in pkhts cells was prevented by supplying PKH2. The t½ values in wild-type cells are similar to published values (9Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 50Zuk D. Jacobson A. EMBO J. 1998; 17: 2914-2925Crossref PubMed Scopus (163) Google Scholar). We conclude from these data that global mRNA decay requires functional Pkh1/2. Deadenylation and decapping are highly regulated steps in mRNA decay (3Parker R. Sheth U. Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 5Goldstrohm A.C. Wickens M. Nat. Rev. Mol. Cell Biol. 2008; 9: 337-344Crossref PubMed Scopus (303) Google Scholar, 7Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (966) Google Scholar, 51Zheng D. Ezzeddine N. Chen C.Y. Zhu W. He X. Shyu A.B. J. Cell Biol. 2008; 182: 89-101Crossref PubMed Scopus (154) Google Scholar, 52Franks T.M. Lykke-Andersen J. Mol. Cell. 2008; 32: 605-615Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). To determine whether Pkh1/2 regulate either step we analyzed a portion of the RNA samples used to generate the data shown in Fig. 1 by using high-resolution polyacrylamide gel electrophoresis (PAGE) (9Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 53Schwartz D.C. Parker R. Mol. Cell. Biol. 1999; 19: 5247-5256Crossref PubMed Scopus (193) Google Scholar). Under these conditions most of the poly(A) tail on MFA2pG mRNA in wild-type cells was hydrolyzed within 8 min or at the rate of about 8 adenosines (A)/min, a rate that remained constant over 2 h (Fig. 2A). Mutant pkhts cells had a similar rate of deadenylation at time 0, but after 30 min at 39 °C the rate dropped to 5.5 A/min and continued to drop over the course of the experiment to a rate of 1.3 A after 2 h. Returning PKH2 to mutant cells restored a normal rate of deadenylation. Similar affects on the rate of deadenylation of endogenous GAL1 mRNA were observed (Fig. 2B). We conclude from these data that Pkh1/2 are required for and regulate the deadenylation step in mRNA decay. Because most functions of Pkh1/2 are mediated by downstream protein kinases Pkc1, Ypk1/2, and Sch9, we determined if any of these kinases control mRNA decay. Analysis of sch9Δ and ypk1ts ypk2Δ mutant cells indicated that Sch9 and Ypk1/2 are not required for mRNA decay (supplemental Fig. S1). Because PKC1 is an essential gene, we examined mRNA decay in a strain with a temperature-sensitive pkc1 allele (54Drgonová J. Drgon T. Roh D.H. Cabib E. J. Cell Biol. 1999; 146: 373-387Crossref PubMed Scopus (48) Google Scholar) using procedures similar to those used with pkhts cells except that pkc1ts cells were incubated at the restrictive temperature of 39 °C for 1 h only before mRNA decay was examined. This shorter incubation time was necessary to avoid cell lysis, which occurs at longer incubation times because the Pkc1 pathway is the master regulator of cell wall maintenance and repair (55Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (303) Google Scholar). We verified experimentally that less than 20% of the cells lysed after 60 min at 39 °C. The half-life of MFA2pG mRNA in pkc1ts cells shifted to the restrictive temperature was 14.2 min, or about 2-fold slower than the 6.4-min half-life observed in wild-type cells (Fig. 3A). The affect of Pkc1 on deadenylation was analyzed by subjecting a portion of RNA to denaturing polyacrylamide gel electrophoresis, which revealed that the reduced rate of decay was due to impaired deadenylation in pkc1ts cells and not to impaired decapping or hydrolysis of the mRNA body in the 5′ to 3′ direction by the XrnI exonuclease (Fig. 3B). For example, in wild-type cells the poly(A) tails were degraded at an average rate of 8.3 A/min, whereas in pkc1ts cells they were degraded at an average rate of 3.8 A/min. Our data are consistent with Pkc1 acting downstream of Pkh1/2 to regulate deadenylation. But to directly verify this hypothesis we sought to show that a constitutively active PKC1 allele (54Drgonová J. Drgon T. Roh D.H. Cabib E. J. Cell Biol. 1999; 146: 373-387Crossref PubMed Scopus (48) Google Scholar) would bypass impaired deadenylation in pkhts mutant cells because such an allele would not require active Pkh. In pkhts mutant cells grown for 1 h at a restrictive temperature the t½ of MFA2pG mRNA was 11.5 min or a little over twice as long as the 4.5 min t½ observed in wild-type cells (Fig. 3C). Mutant cells carrying the constitutive PKC1 allele on a CEN vector had a t½ for MFA2pG mRNA of 7.5 min, showing that the rate of decay was partially restored to the wild-type rate. Likewise, the constitutive PKC1 allele partially restored the rate of deadenylation (Fig. 3D). Partial restoration of the decay and deadenylation rates is expected because cells have both wild-type and constitutive Pkc1 activity. Based upon our data for mRNA decay and deadenylation in pkc1ts cells we conclude that Pkc1 activity is required for and regulates the deadenylation step of the mRNA decay pathway. We determined if the Pkh1/2-Pkc1 pathway controls P-body assembly because the mechanism for controlling their assembly is not known. The Dcp2 subunit of the decapping enzyme, having the green fluorescent protein (GFP) fused to its C terminus, served as a visual reporter for P-body formation (17Sheth U. Parker R. Science. 2003; 300: 805-808Crossref PubMed Scopus (1001) Google Scholar). The pkhts cells were grown at 25 °C, switched to 39 °C for various times, starved 10 min for glucose to induce P-body formation, and examined by fluorescent microscopy. Before the temperature shift (time 0), one or two large P-bodies appeared in wild-type," @default.
• W1984866643 created "2016-06-24" @default.
• W1984866643 creator A5018295855 @default.
• W1984866643 creator A5019944624 @default.
• W1984866643 creator A5023165801 @default.
• W1984866643 creator A5062606338 @default.
• W1984866643 date "2011-03-01" @default.
• W1984866643 modified "2023-10-03" @default.
• W1984866643 title "Nutrients and the Pkh1/2 and Pkc1 Protein Kinases Control mRNA Decay and P-body Assembly in Yeast" @default.
• W1984866643 cites W1484630591 @default.
• W1984866643 cites W1515773506 @default.
• W1984866643 cites W1567346019 @default.
• W1984866643 cites W1849896953 @default.
• W1984866643 cites W1964628768 @default.
• W1984866643 cites W1965722625 @default.
• W1984866643 cites W1971644927 @default.
• W1984866643 cites W1981194277 @default.
• W1984866643 cites W1983788160 @default.
• W1984866643 cites W1991468122 @default.
• W1984866643 cites W2003911773 @default.
• W1984866643 cites W2005767434 @default.
• W1984866643 cites W2016548334 @default.
• W1984866643 cites W2019824435 @default.
• W1984866643 cites W2028583641 @default.
• W1984866643 cites W2030302003 @default.
• W1984866643 cites W2034774838 @default.
• W1984866643 cites W2047255238 @default.
• W1984866643 cites W2052527483 @default.
• W1984866643 cites W2053219805 @default.
• W1984866643 cites W2056644877 @default.
• W1984866643 cites W2057704289 @default.
• W1984866643 cites W2058220225 @default.
• W1984866643 cites W2063678191 @default.
• W1984866643 cites W2070085869 @default.
• W1984866643 cites W2075559286 @default.
• W1984866643 cites W2076627305 @default.
• W1984866643 cites W2079122560 @default.
• W1984866643 cites W2082658854 @default.
• W1984866643 cites W2085519695 @default.
• W1984866643 cites W2088860412 @default.
• W1984866643 cites W2090306001 @default.
• W1984866643 cites W2093611074 @default.
• W1984866643 cites W2096688350 @default.
• W1984866643 cites W2097294952 @default.
• W1984866643 cites W2098337864 @default.
• W1984866643 cites W2101555571 @default.
• W1984866643 cites W2102158431 @default.
• W1984866643 cites W2103724228 @default.
• W1984866643 cites W2104148121 @default.
• W1984866643 cites W2108185386 @default.
• W1984866643 cites W2109550271 @default.
• W1984866643 cites W2112523914 @default.
• W1984866643 cites W2112882175 @default.
• W1984866643 cites W2114717251 @default.
• W1984866643 cites W2114890390 @default.
• W1984866643 cites W2115514152 @default.
• W1984866643 cites W2117403504 @default.
• W1984866643 cites W2117921076 @default.
• W1984866643 cites W2119846936 @default.
• W1984866643 cites W2121100586 @default.
• W1984866643 cites W2122005428 @default.
• W1984866643 cites W2123413580 @default.
• W1984866643 cites W2127743751 @default.
• W1984866643 cites W2129770809 @default.
• W1984866643 cites W2130386199 @default.
• W1984866643 cites W2134530067 @default.
• W1984866643 cites W2136595023 @default.
• W1984866643 cites W2140951250 @default.
• W1984866643 cites W2141325721 @default.
• W1984866643 cites W2143408622 @default.
• W1984866643 cites W2145299132 @default.
• W1984866643 cites W2145853549 @default.
• W1984866643 cites W2146865209 @default.
• W1984866643 cites W2147997023 @default.
• W1984866643 cites W2152571861 @default.
• W1984866643 cites W2152758802 @default.
• W1984866643 cites W2154950250 @default.
• W1984866643 cites W2156435521 @default.
• W1984866643 cites W2157937554 @default.
• W1984866643 cites W2159460045 @default.
• W1984866643 cites W2159990074 @default.
• W1984866643 cites W2163394323 @default.
• W1984866643 cites W2165054897 @default.
• W1984866643 cites W2401949 @default.
• W1984866643 cites W4256110274 @default.
• W1984866643 cites W94107982 @default.
• W1984866643 doi "https://doi.org/10.1074/jbc.m110.196030" @default.
• W1984866643 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3059008" @default.
• W1984866643 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21163942" @default.
• W1984866643 hasPublicationYear "2011" @default.
• W1984866643 type Work @default.
• W1984866643 sameAs 1984866643 @default.
• W1984866643 citedByCount "28" @default.
• W1984866643 countsByYear W19848666432012 @default.
• W1984866643 countsByYear W19848666432013 @default.
• W1984866643 countsByYear W19848666432014 @default.
• W1984866643 countsByYear W19848666432015 @default.
• W1984866643 countsByYear W19848666432016 @default.

• next