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Functional aspects of modified nucleosides in tRNA
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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 [en]
tRNA modification, frameshifting, Elongator complex, Elp1p, Prb1p, proteolysis, glioma, TRM6/61, PKCα
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-109491ISBN: 978-91-7601-355-7 (print)OAI: oai:DiVA.org:umu-109491DiVA: diva2:857303
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
List of papers
1. The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
Open this publication in new window or tab >>The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 19, 9489-9499 p.Article in journal (Refereed) Published
Abstract [en]

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um) nucleosides in the anticodon at the wobble position (U34). Earlier we showed that mutants unable to form the side chain at position 5 (ncm5 or mcm5) or lacking sulphur at position 2 (s2) of U34 result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm5 and s2 side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109852 (URN)10.1093/nar/gkv832 (DOI)000366405600036 ()
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
2. Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells
Open this publication in new window or tab >>Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells
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.

Keyword
Elongator complex, Elp1p, Prb1p, proteolysis, Saccharomyces cerevisiae, tRNA modification
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109853 (URN)10.1002/mbo3.285 (DOI)000368415300002 ()26407534 (PubMedID)
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
3. TRM6/61 connects PKCα with translational control through tRNAiMet stabilization: impact on tumorigenesis
Open this publication in new window or tab >>TRM6/61 connects PKCα with translational control through tRNAiMet stabilization: impact on tumorigenesis
Show others...
2016 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 35, no 14, 1785-1796 p.Article in journal (Refereed) Published
Abstract [en]

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C [alpha] (PKC[alpha]) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAiMet), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAiMet. In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAiMet-overexpressing cells, PKC[alpha] overexpression decreased tRNAiMet expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAiMet expression with decreased expression of PKC[alpha] mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKC[alpha] tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-109854 (URN)10.1038/onc.2015.244 (DOI)000373610400005 ()26234676 (PubMedID)
Note

Supplementary information available for this article at http://www.nature.com/onc/journal/vaop/ncurrent/suppinfo/onc2015244s1.html

Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved

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