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Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells
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).
2015 (English)In: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 4, no 6, 867-878 p.Article in journal (Refereed) Published
Abstract [en]

In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm5) and 5-methylcarboxymethyl (mcm5) side chains on uridines present at the wobble position (U34) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5′ AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.

Place, publisher, year, edition, pages
2015. Vol. 4, no 6, 867-878 p.
Keyword [en]
Elongator complex, Elp1p, Prb1p, proteolysis, Saccharomyces cerevisiae, tRNA modification
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-109853DOI: 10.1002/mbo3.285ISI: 000368415300002PubMedID: 26407534OAI: oai:DiVA.org:umu-109853DiVA: diva2:859495
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Functional aspects of modified nucleosides in tRNA
Open this publication in new window or tab >>Functional aspects of modified nucleosides in tRNA
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity.

First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys.

Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact.

Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 32 p.
Keyword
tRNA modification, frameshifting, Elongator complex, Elp1p, Prb1p, proteolysis, glioma, TRM6/61, PKCα
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109491 (URN)978-91-7601-355-7 (ISBN)
Public defence
2015-10-29, Hörsal D Unod T 9, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-10-08 Created: 2015-09-28 Last updated: 2015-10-08Bibliographically approved

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