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A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase--extensive moonlighting in mitochondrial tRNA biogenesis.

Vilardo E, Nachbagauer C, Buzet A, Taschner A, Holzmann J, Rossmanith W - Nucleic Acids Res. (2012)

Bottom Line: The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes.In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein.Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.

View Article: PubMed Central - PubMed

Affiliation: Center for Anatomy and Cell Biology, Medical University of Vienna, 1090 Vienna, Austria.

ABSTRACT
Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5' extensions, is the methyltransferase responsible for m(1)G9 and m(1)A9 formation. The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.

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TRMT10C has vestigial methyltransferase activity. TRMT10C was tested for tRNA:m1G9 methyltransferase activity at low (40 nM; lane 2) and high concentration (800 nM; lane 3), and at low concentration in the presence of an excess (2 µM) of SDR5C1 (lane 4) or purified as TRMT10C–SDR5C1 complex (lane 6) with different tRNA substrates; (A) (cyt)tRNAArg; (B) (mt)tRNAIle.
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gks910-F7: TRMT10C has vestigial methyltransferase activity. TRMT10C was tested for tRNA:m1G9 methyltransferase activity at low (40 nM; lane 2) and high concentration (800 nM; lane 3), and at low concentration in the presence of an excess (2 µM) of SDR5C1 (lane 4) or purified as TRMT10C–SDR5C1 complex (lane 6) with different tRNA substrates; (A) (cyt)tRNAArg; (B) (mt)tRNAIle.

Mentions: A structural comparison of TRMT10C to its paralogues did not reveal any obvious clue for the requirement of an additional subunit. TRMT10C still seems to be the enzyme’s catalytic subunit, and we sought to determine whether it might have some residual methyltransferase activity in the absence of SDR5C1 under certain conditions. We made use of human cytosolic tRNAArg, which was most efficiently methylated in vitro by all tested enzymes (Figure 5A). A weak m1G spot was detectable after prolonged incubation with high amounts of TRMT10C alone (Figure 7A, lane 3). Under the same conditions, a 20-fold lower amount of TRMT10C accomplished substrate methylation to near completeness if supplemented with an excess of SDR5C1 or pre-associated with it (Figure 7A, lanes 4 and 6). (Mt)tRNAIle, however, was not methylated even by high amounts of TRMT10C alone (Figure 7B), thereby confirming previous findings (Figure 2). TRMT10C, thus, contains a vestigial methyltransferase active site, but it needs to associate with SDR5C1 for efficient catalysis.Figure 7.


A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase--extensive moonlighting in mitochondrial tRNA biogenesis.

Vilardo E, Nachbagauer C, Buzet A, Taschner A, Holzmann J, Rossmanith W - Nucleic Acids Res. (2012)

TRMT10C has vestigial methyltransferase activity. TRMT10C was tested for tRNA:m1G9 methyltransferase activity at low (40 nM; lane 2) and high concentration (800 nM; lane 3), and at low concentration in the presence of an excess (2 µM) of SDR5C1 (lane 4) or purified as TRMT10C–SDR5C1 complex (lane 6) with different tRNA substrates; (A) (cyt)tRNAArg; (B) (mt)tRNAIle.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526285&req=5

gks910-F7: TRMT10C has vestigial methyltransferase activity. TRMT10C was tested for tRNA:m1G9 methyltransferase activity at low (40 nM; lane 2) and high concentration (800 nM; lane 3), and at low concentration in the presence of an excess (2 µM) of SDR5C1 (lane 4) or purified as TRMT10C–SDR5C1 complex (lane 6) with different tRNA substrates; (A) (cyt)tRNAArg; (B) (mt)tRNAIle.
Mentions: A structural comparison of TRMT10C to its paralogues did not reveal any obvious clue for the requirement of an additional subunit. TRMT10C still seems to be the enzyme’s catalytic subunit, and we sought to determine whether it might have some residual methyltransferase activity in the absence of SDR5C1 under certain conditions. We made use of human cytosolic tRNAArg, which was most efficiently methylated in vitro by all tested enzymes (Figure 5A). A weak m1G spot was detectable after prolonged incubation with high amounts of TRMT10C alone (Figure 7A, lane 3). Under the same conditions, a 20-fold lower amount of TRMT10C accomplished substrate methylation to near completeness if supplemented with an excess of SDR5C1 or pre-associated with it (Figure 7A, lanes 4 and 6). (Mt)tRNAIle, however, was not methylated even by high amounts of TRMT10C alone (Figure 7B), thereby confirming previous findings (Figure 2). TRMT10C, thus, contains a vestigial methyltransferase active site, but it needs to associate with SDR5C1 for efficient catalysis.Figure 7.

Bottom Line: The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes.In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein.Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.

View Article: PubMed Central - PubMed

Affiliation: Center for Anatomy and Cell Biology, Medical University of Vienna, 1090 Vienna, Austria.

ABSTRACT
Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5' extensions, is the methyltransferase responsible for m(1)G9 and m(1)A9 formation. The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.

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