Translation of Human β-Actin mRNA is Regulated by mTOR Pathway
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids
2.2. Cell Cultures
2.3. Western Blotting
2.4. DNA Transfection
2.5. NLucP half-life Time Measurement and Luciferase Assay
2.6. Polysome Analysis
2.7. RNA Isolation, cDNA Synthesis and In Vitro Transcription
2.8. 5’RACE
2.9. qPCR
2.10. Statistical Analysis
3. Results
3.1. Translation of Reporter mRNA with Promoter and 5’ UTR of ACTB is mTOR-Sensitive in HEK293T Cells
3.2. Translation of Endogenous ACTB mRNA is Sensitive to Growth Stimulus in HEK293T Cells
3.3. mTOR-Sensitivity of ACTB mRNA Translation is Cell Type-Dependent
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Saxton, R.A.; Sabatini, D.M. mTOR signaling in growth, metabolism, and disease. Cell 2017, 168, 960–976. [Google Scholar] [CrossRef] [PubMed]
- Zoncu, R.; Efeyan, A.; Sabatini, D.M. MTOR: From growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 2011, 12, 21–35. [Google Scholar] [CrossRef]
- Wang, X.; Proud, C.G. MTORC1 signaling: What we still dont know. J. Mol. Cell Biol. 2011, 3, 206–220. [Google Scholar] [CrossRef] [PubMed]
- Thoreen, C.C.; Kang, S.A.; Chang, J.W.; Liu, Q.; Zhang, J.; Gao, Y.; Reichling, L.J.; Sim, T.; Sabatini, D.M.; Gray, N.S. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J. Biol. Chem. 2009, 284, 8023–8032. [Google Scholar] [CrossRef] [PubMed]
- Thoreen, C.C. The molecular basis of mTORC1-regulated translation. Biochem. Soc. Trans. 2017, 45, 213–221. [Google Scholar] [CrossRef]
- Masvidal, L.; Hulea, L.; Furic, L.; Topisirovic, I.; Larsson, O. mTOR-sensitive translation: Cleared fog reveals more trees. RNA Biol. 2017, 14, 1299–1305. [Google Scholar] [CrossRef] [Green Version]
- Meyuhas, O.; Kahan, T. The race to decipher the top secrets of TOP mRNAs. Biochim. Biophys. Acta Gene Regul. Mech. 2015, 1849, 801–811. [Google Scholar] [CrossRef]
- Meyuhas, O. Synthesis of the translational apparatus is regulated at the translational level. Eur. J. Biochem. 2000, 267, 6321–6330. [Google Scholar] [CrossRef] [Green Version]
- Tamarkin-Ben-Harush, A.; Vasseur, J.J.; Debart, F.; Ulitsky, I.; Dikstein, R. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress. eLife 2017, 6, e21907. [Google Scholar] [CrossRef]
- Hsieh, A.C.; Liu, Y.; Edlind, M.P.; Ingolia, N.T.; Janes, M.R.; Sher, A.; Shi, E.Y.; Stumpf, C.R.; Christensen, C.; Bonham, M.J.; et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 2012, 485, 55–61. [Google Scholar] [CrossRef]
- Thoreen, C.C.; Chantranupong, L.; Keys, H.R.; Wang, T.; Gray, N.S.; Sabatini, D.M. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 2012, 485, 109–113. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sinvani, H.; Haimov, O.; Svitkin, Y.; Sonenberg, N.; Tamarkin-Ben-Harush, A.; Viollet, B.; Dikstein, R. Translational tolerance of mitochondrial genes to metabolic energy stress involves TISU and eIF1-eIF4GI cooperation in start codon selection. Cell MeTable 2015, 21, 479–492. [Google Scholar] [CrossRef] [PubMed]
- Gandin, V.; Masvidal, L.; Hulea, L.; Gravel, S.P.; Cargnello, M.; McLaughlan, S.; Cai, Y.; Balanathan, P.; Morita, M.; Rajakumar, A.; et al. NanoCAGE reveals 5’ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs. Genome Res. 2016, 26, 636–648. [Google Scholar] [CrossRef] [PubMed]
- Lyabin, D.N.; Ovchinnikov, L.P. Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein. Sci. Rep. 2016, 6, 22502. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Han, K.; Jaimovich, A.; Dey, G.; Ruggero, D.; Meyuhas, O.; Sonenberg, N.; Meyer, T. Parallel measurement of dynamic changes in translation rates in single cells. Nat. Methods 2014, 11, 86–93. [Google Scholar] [CrossRef]
- Saikia, M.; Wang, X.; Mao, Y.; Wan, J.; Pan, T.; Qian, S.B. Codon optimality controls differential mRNA translation during amino acid starvation. RNA 2016, 22, 1719–1727. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fonseca, B.D.; Zakaria, C.; Jia, J.J.; Graber, T.E.; Svitkin, Y.; Tahmasebi, S.; Healy, D.; Hoang, H.D.; Jensen, J.M.; Diao, I.T.; et al. La-related protein 1 (LARP1) represses terminal oligopyrimidine (TOP) mRNA translation downstream of mTOR complex 1 (mTORC1). J. Biol. Chem. 2015, 290, 15996–16020. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lahr, R.M.; Mack, S.M.; Héroux, A.; Blagden, S.P.; Bousquet-Antonelli, C.; Deragon, J.M.; Berman, A.J. The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5’TOP sequence. Nucleic Acids Res. 2015, 43, 8077–8088. [Google Scholar] [CrossRef] [PubMed]
- Tcherkezian, J.; Cargnello, M.; Romeo, Y.; Huttlin, E.L.; Lavoie, G.; Gygi, S.P.; Roux, P.P. Proteomic analysis of cap-dependent translation identifies LARP1 as a key regulator of 5′TOP mRNA translation. Genes Dev. 2014, 28, 357–371. [Google Scholar] [CrossRef] [PubMed]
- Zhu, J.; Hayakawa, A.; Kakegawa, T.; Kaspar, R.L. Binding of the La autoantigen to the 5′ untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro. Biochim. Biophys. Acta Gene Struct. Exp. 2001, 1521, 19–29. [Google Scholar] [CrossRef]
- Zhu, J.; Spencer, E.D.; Kaspar, R.L. Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes. Biochim. Biophys. Acta Gene Struct. Exp. 2003, 1628, 50–55. [Google Scholar] [CrossRef]
- Yamashita, R.; Suzuki, Y.; Takeuchi, N.; Wakaguri, H.; Ueda, T.; Sugano, S.; Nakai, K. Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics. Nucleic Acids Res. 2008, 36, 3707–3715. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reiter, A.K.; Bolster, D.R.; Crozier, S.J.; Kimball, S.R.; Jefferson, L.S. AMPK represses TOP mRNA translation but not global protein synthesis in liver. Biochem. Biophys. Res. Commun. 2008, 374, 345–350. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dowling, R.J.O.; Topisirovic, I.; Alain, T.; Bidinosti, M.; Fonseca, B.D.; Petroulakis, E.; Wang, X.; Larsson, O.; Selvaraj, A.; Liu, Y.; et al. mTORCI-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 2010, 328, 1172–1176. [Google Scholar] [CrossRef] [PubMed]
- Levy, S.; Avni, D.; Hariharan, N.; Perry, R.P.; Meyuhas, O. Oligopyrimidine tract at the 5’ end of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad. Sci. USA 1991, 88, 3319–3323. [Google Scholar] [CrossRef]
- Biberman, Y.; Meyuhas, O. Substitution of just five nucleotides at and around the transcription start site of rat β-actin promoter is sufficient to render the resulting transcript a subject for translational control. FEBS Lett. 1997, 405, 333–336. [Google Scholar] [CrossRef] [Green Version]
- Tang, H.; Hornstein, E.; Stolovich, M.; Levy, G.; Livingstone, M.; Templeton, D.; Avruch, J.; Meyuhas, O. Amino acid-induced translation of TOP mRNAs Is fully dependent on phosphatidylinositol 3-Kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation. Mol. Cell. Biol. 2001, 21, 8671–8683. [Google Scholar] [CrossRef]
- Morita, M.; Gravel, S.P.; Chénard, V.; Sikström, K.; Zheng, L.; Alain, T.; Gandin, V.; Avizonis, D.; Arguello, M.; Zakaria, C.; et al. MTORC1 controls mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation. Cell MeTable 2013, 18, 698–711. [Google Scholar] [CrossRef]
- Larsson, O.; Morita, M.; Topisirovic, I.; Alain, T.; Blouin, M.-J.; Pollak, M.; Sonenberg, N. Distinct perturbation of the translatome by the antidiabetic drug metformin. Proc. Natl. Acad. Sci. USA 2012, 109, 8977–8982. [Google Scholar] [CrossRef] [Green Version]
- Huo, Y.; Iadevaia, V.; Yao, Z.; Kelly, I.; Cosulich, S.; Guichard, S.; Foster, L.J.; Proud, C.G. Stable isotope-labelling analysis of the impact of inhibition of the mammalian target of rapamycin on protein synthesis. Biochem. J. 2012, 444, 141–151. [Google Scholar] [CrossRef] [Green Version]
- Livingstone, M.; Sikström, K.; Robert, P.A.; Uzé, G.; Larsson, O.; Pellegrini, S.P. Assessment of mTOR-dependent translational regulation of interferon stimulated genes. PLoS ONE 2015, 10, e0133482. [Google Scholar] [CrossRef] [PubMed]
- Forrest, A.R.R.; Kawaji, H.; Rehli, M.; Baillie, J.K.; de Hoon, M.J.L.; Lassmann, T.; Itoh, M.; Summers, K.M.; Suzuki, H.; Daub, C.O.; et al. A promoter-level mammalian expression atlas. Nature 2014, 507, 462–470. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Suzuki, A.; Kawano, S.; Mitsuyama, T.; Suyama, M.; Kanai, Y.; Shirahige, K.; Sasaki, H.; Tokunaga, K.; Tsuchihara, K.; Sugano, S.; et al. DBTSS/DBKERO for integrated analysis of transcriptional regulation. Nucleic Acids Res. 2018, 46, D229–D238. [Google Scholar] [CrossRef]
- Lyabin, D.N.; Eliseeva, I.A.; Ovchinnikov, L.P. YB-1 Synthesis is regulated by mTOR signaling pathway. PLoS ONE 2012, 7, e52527. [Google Scholar] [CrossRef] [PubMed]
- Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10. [Google Scholar] [CrossRef]
- Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10, R25. [Google Scholar] [CrossRef] [PubMed]
- Quinlan, A.R. BEDTools: The Swiss-Army tool for genome feature analysis. Curr. Protoc. Bioinform. 2014, 47, 11.12.1–11.12.34. [Google Scholar] [CrossRef] [PubMed]
- Thorne, N.; Inglese, J.; Auld, D.S. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem. Biol. 2010, 17, 646–657. [Google Scholar] [CrossRef]
- Boisvert, F.-M.; Ahmad, Y.; Gierliński, M.; Charrière, F.; Lamont, D.; Scott, M.; Barton, G.; Lamond, A.I. A Quantitative spatial proteomics analysis of proteome turnover in human cells. Mol. Cell. Proteomics 2012, 11, 1429. [Google Scholar] [CrossRef]
- Kleene, K.C.; Cataldo, L.; Mastrangelo, M.-A.A.; Tagne, J.-B.B. Alternative patterns of transcription and translation of the ribosomal protein L32 mRNA in somatic and spermatogenic cells in mice. Exp. Cell Res. 2003, 291, 101–110. [Google Scholar] [CrossRef]
- Eliseeva, I.; Vorontsov, I.; Babeyev, K.; Buyanova, S.; Sysoeva, M.; Kondrashov, F.; Kulakovskiy, I. In silico motif analysis suggests an interplay of transcriptional and translational control in mTOR response. Translation 2013, 1, 18–24. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Avni, D.; Shama, S.; Loreni, F.; Meyuhas, O. Vertebrate mRNAs with a 5’-terminal pyrimidine tract are candidates for translational repression in quiescent cells: Characterization of the translational cis-regulatory element. Mol. Cell. Biol. 1994, 14, 3822–3833. [Google Scholar] [CrossRef] [PubMed]
- Jefferies, H.B.J.; Thomas, G.; Thomas, G. Elongation factor-1α mRNA is selectively translated following mitogenic stimulation. J. Biol. Chem. 1994, 269, 4367–4372. [Google Scholar] [PubMed]
- Gao, X.; Wan, J.; Liu, B.; Ma, M.; Shen, B.; Qian, S.B. Quantitative profiling of initiating ribosomes in vivo. Nat. Methods 2015, 12, 147–153. [Google Scholar] [CrossRef] [PubMed]
- Grolleau, A.; Bowman, J.; Pradet-Balade, B.; Puravs, E.; Hanash, S.; Garcia-Sanz, J.A.; Beretta, L. Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics. J. Biol. Chem. 2002, 277, 22175–22184. [Google Scholar] [CrossRef] [PubMed]
- Morita, T.; Sobuě, K. Specification of neuronal polarity regulated by local translation of CRMP2 and tau via the mTOR-p70S6K pathway. J. Biol. Chem. 2009, 284, 27734–27745. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Z.C.; Nechushtan, H.; Jacob-Hirsch, J.; Avni, D.; Meyuhas, O.; Razin, E. Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells. Oncogene 1998, 16, 763–769. [Google Scholar] [CrossRef] [Green Version]
- Hagner, P.R.; Mazan-Mamczarz, K.; Dai, B.; Balzer, E.M.; Corl, S.; Martin, S.S.; Zhao, X.F.; Gartenhaus, R.B. Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5′ terminal oligopyrimidine tract. Oncogene 2011, 30, 1531–1541. [Google Scholar] [CrossRef]
- Avni, D.; Biberman, Y.; Meyuhas, O. The 5′ terminal oligopyrimidine tract confers translational control on TOP mRNAs in a cell type-and sequence context-dependent manner. Nucleic Acids Res. 1996, 25, 995–1001. [Google Scholar] [CrossRef]
- Choo, A.Y.; Yoon, S.-O.; Kim, S.G.; Roux, P.P.; Blenis, J. Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc. Natl. Acad. Sci. USA 2008, 105, 17414–17419. [Google Scholar] [CrossRef] [Green Version]
- Zid, B.M.; O’Shea, E.K. Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast. Nature 2014, 514, 117–121. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bregman, A.; Avraham-Kelbert, M.; Barkai, O.; Duek, L.; Guterman, A.; Choder, M. Promoter elements regulate cytoplasmic mRNA decay. Cell 2011, 147, 1473–1483. [Google Scholar] [CrossRef] [PubMed]
- Slobodin, B.; Han, R.; Calderone, V.; Vrielink, J.A.F.O.; Loayza-Puch, F.; Elkon, R.; Agami, R. Transcription impacts the efficiency of mRNA translation via co-transcriptional N6-adenosine methylation. Cell 2017, 169, 326–337.e12. [Google Scholar] [CrossRef] [PubMed]
- Parry, T.J.; Theisen, J.W.M.; Hsu, J.Y.; Wang, Y.L.; Corcoran, D.L.; Eustice, M.; Ohler, U.; Kadonaga, J.T. The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery. Genes Dev. 2010, 24, 2013–2018. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Coots, R.A.; Liu, X.-M.; Mao, Y.; Dong, L.; Zhou, J.; Wan, J.; Zhang, X.; Qian, S.-B. m6A Facilitates eIF4F-independent mRNA Translation. Mol. Cell 2017, 68, 504–514.e7. [Google Scholar] [CrossRef] [PubMed]
- Meyer, K.D.; Saletore, Y.; Zumbo, P.; Elemento, O.; Mason, C.E.; Jaffrey, S.R. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 2012, 149, 1635–1646. [Google Scholar] [CrossRef]
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Eliseeva, I.; Vasilieva, M.; Ovchinnikov, L.P. Translation of Human β-Actin mRNA is Regulated by mTOR Pathway. Genes 2019, 10, 96. https://doi.org/10.3390/genes10020096
Eliseeva I, Vasilieva M, Ovchinnikov LP. Translation of Human β-Actin mRNA is Regulated by mTOR Pathway. Genes. 2019; 10(2):96. https://doi.org/10.3390/genes10020096
Chicago/Turabian StyleEliseeva, Irina, Maria Vasilieva, and Lev P. Ovchinnikov. 2019. "Translation of Human β-Actin mRNA is Regulated by mTOR Pathway" Genes 10, no. 2: 96. https://doi.org/10.3390/genes10020096
APA StyleEliseeva, I., Vasilieva, M., & Ovchinnikov, L. P. (2019). Translation of Human β-Actin mRNA is Regulated by mTOR Pathway. Genes, 10(2), 96. https://doi.org/10.3390/genes10020096