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  • 1. Bento-Abreu, Andre
    et al.
    Jager, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Swinnen, Bart
    Rué, Laura
    Hendrickx, Stijn
    Jones, Ashley
    Staats, Kim A.
    Taes, Ines
    Eykens, Caroline
    Nonneman, Annelies
    Nuyts, Rik
    Timmers, Mieke
    Silva, Lara
    Chariot, Alain
    Nguyen, Laurent
    Ravits, John
    Lemmens, Robin
    Cabooter, Deirdre
    Van Den Bosch, Ludo
    Van Damme, Philip
    Al-Chalabi, Ammar
    Bystrom, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Robberecht, Wim
    Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.2018In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 27, no 7, p. 1276-1289Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

  • 2.
    Björk, Glenn
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.2007In: RNA, ISSN 1355-8382, Vol. 13, no 8, p. 1245-55Article in journal (Refereed)
    Abstract [en]

    Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.

  • 3.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Jacobsson, Kerstin
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A primordial tRNA modification required for the evolution of life?2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 1-2, p. 231-239Article in journal (Refereed)
    Abstract [en]

    The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

  • 4.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wikström, P M
    Byström, Anders S
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine1989In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 244, no 4907, p. 986-989Article in journal (Refereed)
    Abstract [en]

    The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

  • 5.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12.1982In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 188, no 3, p. 440-446Article in journal (Refereed)
    Abstract [en]

    The trmD gene, which governs the formation of 1-methyl-guanosine(m1G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA+ hybrid plasmid was isolated, which carried the trmD+ gene and was shown to over-produce the tRNA(m1G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD+ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA+ gene. The mutation trmD1, which renders the tRNA(m1G)methyltransferase temperature-sensitive both in vivo and in vitro could be complemented by trmD+ plasmids. These results suggest that the gene trmD+ is the structural gene for the tRNA(m1G)methyltransferase (EC 2.1.1.3.1).

  • 6.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-121982In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 188, no 3, p. 447-454Article in journal (Refereed)
    Abstract [en]

    The trmD gene, which is the structural gene for the tRNA(m1G)-methyltransferase, is shown to be part of a polycistronic operon. A 4.6 kb SalI-EcoRI chromosomal DNA fragment contains the trmD gene (Byström and Björk 1982). Subclonings, deletion mapping and Tn5 insertions into plasmid pBY03 have established the gene organization of the trmD area on the Escherichia coli chromosome. The different plasmid derivatives were analysed for expression of protein products using the minicell system. Such analyses established the organisation of genes encoding six polypeptides to be SalI1-48 K-13 K-25 K-31 K-15 K-16 K-EcoRI1. The 31 K polypeptide was shown to be the tRNA(m1G)methyltransferase. The trmD operon encodes for four polypeptides; 13 K-25 K-31 K(trmD)-15 K and the direction of transcription is from 13 K (promoter proximal) to 15 K (promoter distal). However, there might be a weak internal promoter between the trmD gene and the gene encoding the 15 K product. The level of expression from this operon in the minicell system does not seem to follow normal polarity since we observed high expression of 13 K, 25 K, and 15 K products but low expression of the internal trmD gene.

  • 7.
    Byström, Anders S.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fink, G. R.
    A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast1989In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 216, no 2-3, p. 276-286Article in journal (Refereed)
    Abstract [en]

    Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA(IMet). Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA(IMet) is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slow; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.

  • 8.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hjalmarsson, Karin J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide1983In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 2, no 6, p. 899-905Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.

  • 9.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Gabain, A
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species1989In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 208, no 4, p. 575-586Article in journal (Refereed)
    Abstract [en]

    The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

  • 10.
    Chapman, K B
    et al.
    Umeå University, Faculty of Medicine, Microbiology. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Boeke, J D
    Initiator methionine tRNA is essential for Ty1 transposition in yeast1992In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 89, no 8, p. 3236-3240Article in journal (Refereed)
    Abstract [en]

    The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

  • 11.
    Chen, Changchun
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Anderson, James T
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Unexpected accumulation of ncm5U and ncm5s2U in a trm9 mutant suggests an additional step in the synthesis of mcm5U and mcm5s2U.2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e20783-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm(5)U), 5-carbamoylmethyl-2'-O-methyluridine (ncm(5)Um), 5-methoxycarbonylmethyl-uridine (mcm(5)U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The formation of mcm(5) and ncm(5) side chains involves a complex pathway, where the last step in formation of mcm(5) is a methyl esterification of cm(5) dependent on the Trm9 and Trm112 proteins.

    METHODOLOGY AND PRINCIPAL FINDINGS: Both Trm9 and Trm112 are required for the last step in formation of mcm(5) side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm(5)U or mcm(5)s(2)U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm(5)U and mcm(5)s(2)U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm(5)U and ncm(5)s(2)U, not the expected cm(5)U and cm(5)s(2)U.

    CONCLUSIONS: Trm9p and Trm112p function together at the final step in formation of mcm(5)U in tRNA by using the intermediate cm(5)U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm(5)U and ncm(5)s(2)U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm(5) side chain. We propose two alternative explanations for this observation. One is that the intermediate cm(5)U is generated from ncm(5)U by a yet unknown mechanism and the other is that cm(5)U is formed before ncm(5)U and mcm(5)U.

  • 12.
    Chen, Changchun
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Eliasson, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rydén, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elongator Complex Influences Telomeric Gene Silencing and DNA Damage Response by Its Role in Wobble Uridine tRNA Modification2011In: PLoS genetics, ISSN 1553-7404, Vol. 7, no 9, p. e1002258-Article in journal (Refereed)
    Abstract [en]

    Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm(5)U(34)), 5-methoxycarbonylmethyluridine (mcm(5)U(34)), and 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys) (s(2) ) (UUU), tRNA(Gln) (s(2) ) (UUG), and tRNA(Glu) (s(2) ) (UUC), which in a wild-type background contain the mcm(5)s(2)U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U(34). Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm(5)s(2)U nucleoside in tRNA(Lys) (mcm(5) (s(2) ) (UUU) ), tRNA(Gln) (mcm(5) (s(2) ) (UUG) ), and tRNA(Glu) (mcm(5) (s(2) ) (UUC) ). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U(34) are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

  • 13.
    Chen, Changchun
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tuck, Simon
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Byström, Anders S
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants2009In: PLoS genetics, ISSN 1553-7404, Vol. 5, no 7, p. e1000561-Article in journal (Refereed)
    Abstract [en]

    Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5'methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development.

  • 14. Dever, Thomas E
    et al.
    Yang, Weimin
    Åström, Stefan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Byström, Anders S
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hinnebusch, A G
    Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes1995In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 15, no 11, p. 6351-6363Article in journal (Refereed)
    Abstract [en]

    To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.

  • 15.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.2006In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 24, no 1, p. 139-148Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis, and formation of modified wobble uridines in tRNA. Therefore it was important to clarify whether Elongator has three distinct functions or whether it regulates one key process that leads to multiple downstream effects. Here, we show that the phenotypes of Elongator-deficient cells linking the complex to transcription and exocytosis are suppressed by increased expression of two tRNA species. Elongator is required for formation of the mcm(5) group of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) in these tRNAs. Hence, in cells with normal levels of these tRNAs, presence of mcm(5)s(2)U is crucial for posttranscriptional expression of gene products important in transcription and exocytosis. Our results indicate that the physiologically relevant function of the evolutionary-conserved Elongator complex is in formation of modified nucleosides in tRNAs.

  • 16.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moqtaderi, Zarmik
    Fan, Xiaochun
    Lu, Jian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Struhl, Kevin
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Iwr1 protein is important for preinitiation complex formation by all three nuclear RNA polymerases in Saccharomyces cerevisiae2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e20829-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II.

    PRINCIPAL FINDINGS: Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect.

    CONCLUSIONS: Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.

  • 17. Friant, S
    et al.
    Heyman, T
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wilhelm, M
    Wilhelm, F X
    Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo1998In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 18, no 2, p. 799-806Article in journal (Refereed)
    Abstract [en]

    Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).

  • 18.
    Hjalmarsson, Karin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli1983In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 258, no 2, p. 1343-1351Article in journal (Refereed)
    Abstract [en]

    The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

  • 19.
    Huang, Bo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J.O.
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An early step in wobble uridine tRNA modification requires the Elongator complex2005In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 4, p. 424-436Article in journal (Refereed)
    Abstract [en]

    Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1–ELP6) genes or the three killer toxin insensitivity (KTI11–KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1–ELP6 and KTI11–KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1–elp6 and kti11–kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1–ELP6 or KTI11KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

  • 20. Huang, Bo
    et al.
    Lu, Jian
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae.2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 10, p. 2183-2194Article in journal (Refereed)
    Abstract [en]

    We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.

  • 21. Johansson, Marcus J O
    et al.
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA.2004In: RNA, ISSN 1355-8382, Vol. 10, no 4, p. 712-9Article in journal (Refereed)
    Abstract [en]

    The biogenesis of transfer RNA is a process that requires many different factors. In this study, we describe a genetic screen aimed to identify gene products participating in this process. By screening for mutations lethal in combination with a sup61-T47:2C allele, coding for a mutant form of, the nonessential TAN1 gene was identified. We show that the TAN1 gene product is required for formation of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA. In Saccharomyces cerevisiae, ac(4)C is present at position 12 in tRNAs specific for leucine and serine as well as in 18S ribosomal RNA. Analysis of RNA isolated from a tan1-null mutant revealed that ac(4)C was absent in tRNA, but not rRNA. Although no tRNA acetyltransferase activity by a GST-Tan1 fusion protein was detected, a gel-shift assay revealed that Tan1p binds tRNA, suggesting a direct role in synthesis of ac(4)C(12). The absence of the TAN1 gene in the sup61-T47:2C mutant caused a decreased level of mature, indicating that ac(4)C(12) and/or Tan1p is important for tRNA stability.

  • 22.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation2002In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 8, no 3, p. 324-335Article in journal (Refereed)
    Abstract [en]

    A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

  • 23.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Transfer RNA modifications and modifying enzymes in Saccharomyces cerevisiae.2005In: Fine-Tuning of RNA Functions by Modification and Editing. / [ed] Henri Grosjean, Springer Berlin/Heidelberg, 2005, p. 87-120Chapter in book (Refereed)
    Abstract [en]

    Transfer RNAs are adaptor molecules, which decode mRNA into protein and, thereby, play a central role in gene expression. During the maturation of a primary tRNA transcript, specific subsets of the four normal nucleosides adenosine, cytidine, guanosine, and uridine are modified. The formation of a modified nucleoside can require more than one gene product and may involve several enzymatic steps. In the last few years, the identification of gene products required for formation of modified nucleosides in tRNA has dramatically increased. In this review, proteins involved in modification of cytoplasmic tRNAs in Saccharomyces cerevisiae are described, emphasizing phenotypic characteristics of modification deficient strains and genetic approaches used to determine the in vivo role of modified nucleosides/modifying enzymes.

  • 24.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 10, p. 3301-3312Article in journal (Refereed)
    Abstract [en]

    The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side chain and sometimes an additional 2-thio (s2) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

  • 25.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Xu, Fu
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elongator-a tRNA modifying complex that promotes efficient translational decoding2018In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1861, no 4, p. 401-408Article in journal (Refereed)
    Abstract [en]

    Naturally occurring modifications of the nucleosides in the anticodon region of tRNAs influence their translational decoding properties. Uridines present at the wobble position in eukaryotic cytoplasmic tRNAs often contain a 5-carbamoylmethyl (ncm5) or 5-methoxycarbonylmethyl (mcm5) side-chain and sometimes also a 2-thio or 2'-O-methyl group. The first step in the formation of the ncm5 and mcm5 side-chains requires the conserved six-subunit Elongator complex. Although Elongator has been implicated in several different cellular processes, accumulating evidence suggests that its primary, and possibly only, cellular function is to promote modification of tRNAs. In this review, we discuss the biosynthesis and function of modified wobble uridines in eukaryotic cytoplasmic tRNAs, focusing on the in vivo role of Elongator-dependent modifications in Saccharomyces cerevisiae. 

  • 26.
    Karlsborn, Tony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mahmud, A K M Firoj
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tükenmez, Hasan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile2016In: Metabolomics, ISSN 1573-3882, E-ISSN 1573-3890, Vol. 12, no 12, article id 177Article in journal (Refereed)
    Abstract [en]

    Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm(5)) and 5-methoxycarbonylmethyl (mcm(5)) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu), which in wild-type strains are modified with mcm(5)s(2)U, partially suppress phenotypes of an elp3 Delta strain. Objectives: To identify metabolic alterations in an elp3 Delta strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3 Delta strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). The temperature sensitive elp3 Delta strain derivatives were cultivated at permissive (30 degrees C) or semi-permissive (34 degrees C) growth conditions. Results: Culturing an elp3 Delta strain at 30 or 34 degrees C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3D strain carrying the wild-type ELP3 gene. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu) suppressed a subset of the metabolic alterations observed in the elp3 Delta strain. Conclusion: Our results suggest that the presence of ncm(5)- and mcm(5)-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

  • 27.
    Karlsborn, Tony
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tukenmez, Hasan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Chen, Changchun
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcm(5)s(2)U in tRNA2014In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 454, no 3, p. 441-445Article in journal (Refereed)
    Abstract [en]

    Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides. 

  • 28.
    Karlsborn, Tony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tukenmez, Hasan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mahmud, A. K. M. Firoj
    Xu, Fu
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Xu, Hao
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes2014In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, no 12, p. 1519-1528Article in journal (Refereed)
    Abstract [en]

    Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U-34 and their influence on Elongator activity.

  • 29. Karlsson, Roger
    et al.
    Aspenström, Pontus
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene1991In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 11, no 1, p. 213-217Article in journal (Refereed)
    Abstract [en]

    Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.

  • 30. Keeney, Jill B
    et al.
    Chapman, Karen B
    Lauermann, Vit
    Voytas, Daniel F
    Åström, Stefan U
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Boeke, Jeff D
    Multiple molecular determinants for retrotransposition in a primer tRNA1995In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 15, no 1, p. 217-226Article in journal (Refereed)
    Abstract [en]

    Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

  • 31. Kim, Sunhong
    et al.
    Johnson, Wade
    Chen, Changchun
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sewell, Aileen K
    Byström, Anders S
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Han, Min
    Allele-specific suppressors of lin-1(R175Opal) identify functions of MOC-3 and DPH-3 in tRNA modification complexes in Caenorhabditis elegans2010In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 185, no 4, p. 1235-1247Article in journal (Refereed)
    Abstract [en]

    The elongator (ELP) complex consisting of Elp1-6p has been indicated to play roles in multiple cellular processes. In yeast, the ELP complex has been shown to genetically interact with Uba4p/Urm1p and Kti11-13p for a function in tRNA modification. Through a Caenorhabditis elegans genetic suppressor screen and positional cloning, we discovered that loss-of-function mutations of moc-3 and dph-3, orthologs of the yeast UBA4 and KTI11, respectively, effectively suppress the Multivulva (Muv) phenotype of the lin-1(e1275, R175Opal) mutation. These mutations do not suppress the Muv phenotype caused by other lin-1 alleles or by gain-of-function alleles of ras or raf that act upstream of lin-1. The suppression can also be reverted by RNA interference of lin-1. Furthermore, we showed that dph-3(lf) also suppressed the defect of lin-1(e1275) in promoting the expression of a downstream target (egl-17). These results indicate that suppression by the moc-3 and dph-3 mutations is due to the elevated activity of lin-1(e1275) itself rather than the altered activity of a factor downstream of lin-1. We further showed that loss-of-function mutations of urm-1 and elpc-1-4, the worm counterparts of URM1 and ELP complex components in yeast, also suppressed lin-1(e1275). We also confirmed that moc-3(lf) and dph-3(lf) have defects in tRNA modifications as do the mutants of their yeast orthologs. These results, together with the observation of a likely readthrough product from a lin-1(e1275)∷gfp fusion transgene indicate that the aberrant tRNA modification led to failed recognition of a premature stop codon in lin-1(e1275). Our genetic data suggest that the functional interaction of moc-3/urm-1 and dph-3 with the ELP complex is an evolutionarily conserved mechanism involved in tRNA functions that are important for accurate translation.

  • 32. Korch, C
    et al.
    Mountain, H A
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae1991In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 229, no 1, p. 96-108Article in journal (Refereed)
    Abstract [en]

    The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP:adenylylsulfate-3'-phosphotransferase, EC 2.7.1.25), has been cloned. The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23,060 dalton. Translational fusions of MET14 with the beta-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca. 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium. By primer extension the MET14 transcripts were found to start between positions -25 and -45 upstream of the initiator codon. Located upstream of the MET14 gene is a perfect match (positions -222 to -229) with the previously proposed methionine-specific upstream activating sequence (UASMet). This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PHO5 gene to which the yeast protein PHO4 binds. The human oncogenic protein c-Myc also has the same recognition sequence. Furthermore, in the 270 bp upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element. The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E. coli and chromosomally integrated. We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively. We propose that both types of control are exerted on MET14.

  • 33. Koshla, Oksana
    et al.
    Yushchuk, Oleksandr
    Stash, Iryna
    Dacyuk, Yuriy
    Myronovskyi, Maksym
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sussmuth, Roderich D.
    Luzhetskyy, Andriy
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kirsebom, Leif A.
    Ostash, Bohdan
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, no 1, p. 249-265Article in journal (Refereed)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 34. Leitner, Johannes
    et al.
    Retzer, Katarzyna
    Malenica, Nenad
    Bartkeviciute, Rasa
    Lucyshyn, Doris
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Korbei, Barbara
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Luschnig, Christian
    Meta-regulation of Arabidopsis Auxin Responses Depends on tRNA Maturation2015In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 11, no 4, p. 516-526Article in journal (Refereed)
    Abstract [en]

    Polar transport of the phytohormone auxin throughout plants shapes morphogenesis and is subject to stringent and specific control. Here, we identify basic cellular activities connected to translational control of gene expression as sufficient to specify auxin-mediated development. Mutants in subunits of Arabidopsis Elongator, a protein complex modulating translational efficiency via maturation of tRNAs, exhibit defects in auxin-controlled developmental processes, associated with reduced abundance of PIN-formed (PIN) auxin transport proteins. Similar anomalies are observed upon interference with tRNA splicing by downregulation of RNA ligase (AtRNL), pointing to a general role of tRNA maturation in auxin signaling. Elongator Protein 6 (ELP6) and AtRNL expression patterns underline an involvement in adjusting PIN protein levels, whereas rescue of mutant defects by auxin indicates rate-limiting activities in auxin-controlled organogenesis. This emphasizes mechanisms in which auxin serves as a bottleneck for plant morphogenesis, translating common cellular activities into defined developmental readouts.

  • 35.
    Lu, Jian
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Identification of determinants in tRNA required for Kluyveromyces lactis γ-toxin cleavageManuscript (Other academic)
  • 36. Lu, Jian
    et al.
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kluyveromyces lactis γ-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 4, p. 1072-1080Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis gamma-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae [see text] between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) residue at position 34 (wobble position) of which the mcm(5) group is required for efficient cleavage. However, the different cleavage efficiencies of mcm(5)s(2)U(34)-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by gamma-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate [see text] is cleaved at the same position as the natural substrate. In [see text], the nucleotides U(34)U(35)C(36)A(37)C(38) are required for optimal gamma-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast [see text] reinforced the strong stimulatory effects of the mcm(5) group, revealed a weak positive effect of the s(2) group and a negative effect of the bacterial 5-methylaminomethyl (mnm(5)) group. The data underscore the high specificity of this yeast tRNA toxin.

  • 37.
    Lu, Jian
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridineManuscript (Other (popular science, discussion, etc.))
  • 38.
    Lu, Jian
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Kluyveromyces lactis γ-toxin targets tRNA anticodons.2005In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 11, p. 1648-1654Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

  • 39. Macari, F.
    et al.
    El-houfi, Y.
    Boldina, G.
    Xu, Hao
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Khoury-Hanna, S.
    Ollier, J.
    Yazdani, L.
    Zheng, G.
    Bieche, I.
    Legrand, N.
    Paulet, D.
    Durrieu, S.
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Delbecq, S.
    Lapeyre, B.
    Bauchet, L.
    Pannequin, J.
    Hollande, F.
    Pan, T.
    Teichmann, M.
    Vagner, S.
    David, A.
    Choquet, A.
    Joubert, D.
    TRM6/61 connects PKCα with translational control through tRNAiMet stabilization: impact on tumorigenesis2016In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 35, no 14, p. 1785-1796Article in journal (Refereed)
    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.

  • 40. Mehlgarten, Constance
    et al.
    Jablonowski, Daniel
    Wrackmeyer, Uta
    Tschitschmann, Susan
    Sondermann, David
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gong, Zhizhong
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Schaffrath, Raffael
    Breunig, Karin D
    Elongator function in tRNA wobble uridine modification is conserved between yeast and plants2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, no 5, p. 1082-1094Article in journal (Refereed)
    Abstract [en]

    Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator’s tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast-plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.

  • 41. Mountain, H A
    et al.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Korch, C
    The general amino acid control regulates MET4, which encodes a methionine-pathway-specific transcriptional activator of Saccharomyces cerevisiae1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 7, no 2, p. 215-228Article in journal (Refereed)
    Abstract [en]

    A met4 mutant of Saccharomyces cerevisiae was unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway. The sequence of the cloned MET4 gene allowed the previously sequenced flanking LEU4 and POL1 genes to be linked to MET4 into a 10,327 bp contiguous region of chromosome XIV. From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C-terminus, preceded by both acidic and basic regions. Thus, MET4 belongs to the family of basic leucine zipper trans-activator proteins. Disruption of MET4 resulted in methionine auxotrophy with no other phenotype. Transcriptional studies showed that MET4 was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4. GCN4 binding sequences are present between the divergently transcribed MET4 and LEU4 genes. Over-expression of MET4 resulted in leaky expression from the otherwise tightly regulated MET3 promoter under its control. The presence of consensus sequences for other potential regulatory elements in the MET4 promoter suggests a complex regulation of this gene.

  • 42. Mountain, Harry A
    et al.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsen, J T
    Korch, C
    Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae1991In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 7, no 8, p. 781-803Article in journal (Refereed)
    Abstract [en]

    Genes encoding enzymes in the threonine/methionine biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.

  • 43.
    Nordlund, Monica E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, J O Marcus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.2000In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 6, no 6, p. 844-60Article in journal (Refereed)
    Abstract [en]

    The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

  • 44.
    Tükenmez, Hasan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Magnussen, Helge
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 9, article id e0163115Article in journal (Refereed)
    Abstract [en]

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies.

  • 45.
    Tükenmez, Hasan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Xu, Hao
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 19, p. 9489-9499Article in journal (Refereed)
    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.

  • 46.
    von Pawel-Rammingen, Ulrich
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åström, Stefan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae1992In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 12, no 4, p. 1432-1442Article in journal (Refereed)
    Abstract [en]

    The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.

  • 47.
    Wikström, P M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli1988In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 203, no 1, p. 141-152Article in journal (Refereed)
    Abstract [en]

    The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

  • 48.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications2019In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 8, article id e1008117Article in journal (Refereed)
    Abstract [en]

    The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5 ) and 5-carbamoylmethyl (ncm5 ) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5 /ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5 /ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2- thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.

  • 49.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Zhou, Yang
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of factors that promote biogenesis of tRNACGASer.2018In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1286-1294Article in journal (Refereed)
    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.

  • 50.
    Xu, Hao
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bygdell, Joakim
    Wingsle, Gunnar
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells2015In: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 4, no 6, p. 867-878Article in journal (Refereed)
    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.

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