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Identification of factors that promote biogenesis of tRNACGASer
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).ORCID iD: 0000-0001-7117-7426
2018 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1286-1294Article in journal (Refereed) Published
Abstract [en]

A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of tRNACGASer. Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and Bud13 as a factor controlling the levels of ac4C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) tRNACGASer. Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered tRNACGASer. These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence tRNACGASer biogenesis.

Place, publisher, year, edition, pages
Taylor & Francis, 2018. Vol. 15, no 10, p. 1286-1294
Keywords [en]
modified nucleosides, sup61, tRNA maturation, tRNA modification, tRNASer
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-154010DOI: 10.1080/15476286.2018.1526539ISI: 000450608900004PubMedID: 30269676OAI: oai:DiVA.org:umu-154010DiVA, id: diva2:1269868
Funder
Magnus Bergvall Foundation, 2017-02098Swedish Research Council, 621-2016-03949Available from: 2018-12-11 Created: 2018-12-11 Last updated: 2019-12-16Bibliographically approved
In thesis
1. Factors modulating tRNA biogenesis and function in Saccharomyces cerevisiae
Open this publication in new window or tab >>Factors modulating tRNA biogenesis and function in Saccharomyces cerevisiae
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNA (tRNA) genes are transcribed by RNA polymerase III as precursors that undergo multiple processing steps to form mature tRNAs. These steps include processing of the 5’ leader and 3’ trailer sequences, addition of a 3’ CCA tail, removal of introns, and formation of modified nucleosides. The mature tRNAs carry amino acids to the ribosome where proteins are synthesized. The aim of this thesis is to identify and characterize factors that influence tRNA biogenesis and function in Saccharomyces cerevisiae.

Nonsense suppressor tRNAs are encoded by mutated tRNA genes and able to read stop codons. The SUP4 gene encodes such a suppressor tRNA that base-pairs with UAA stop codons. By screening for mutations that impair the nonsense suppression of the SUP4-encoded tRNA, we identified a loss-of-function mutation in the YPK9 gene. Inactivation of Ypk9p causes a reduction in the readthrough of UAA stop codon. We found that phenotypes of ypk9Δ cells including decreased UAA readthrough and sensitivity to Mn2+ are counteracted by increasing the cellular levels of putrescine, one type of polyamine. Importantly, cells lacking Ypk9p show reduced levels of putrescine. Our results suggest that the YPK9 gene product influences the cellular levels of putrescine, which plays a role in maintaining the fidelity of translation termination.

The Elongator complex, consisting of Elp1p-Elp6p six proteins, catalyzes the formation of U34 modifications in the anticodon region of 11 tRNA species. Elongator mutants display pleiotropic phenotypes that are caused by decreased tRNA functionality. We found that the genetic background, largely due to a polymorphism at the SSD1 locus, influences the pleiotropic phenotypes of Elongator mutants.

In a genetic screen for factors that are essential for the survival of cells encoding a destabilized tRNASerCGA, several gene products were identified. We demonstrate that mutations in these genes result in reduced levels of the destabilized tRNASerCGA, suggesting a role for these gene products in tRNASerCGA biosynthesis.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2019. p. 43
Keywords
Translation, tRNA biogenesis, suppressor tRNA, Elongator complex, wobble uridine modifications, SUP4, YPK9, SSD1, serine tRNA, synthetic lethality, yeast
National Category
Biological Sciences Genetics
Research subject
biology; Molecular Biology; Genetics
Identifiers
urn:nbn:se:umu:diva-166141 (URN)978-91-7855-167-5 (ISBN)978-91-7855-168-2 (ISBN)
Public defence
2019-01-29, Naturvetarhuset, N320, Umeå, 09:30 (English)
Opponent
Supervisors
Available from: 2019-12-20 Created: 2019-12-12 Last updated: 2019-12-17Bibliographically approved

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Xu, FuZhou, YangByström, AndersJohansson, Marcus J. O.

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