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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 is the subunit primarily responsible for tRNA binding. (A and B) Labelled pre-tRNAs were incubated with increasing concentrations of SDR5C1, TRMT10C or the TRMT10C–SDR5C1 complex and were resolved by native PAGE; representative gels are shown. The concentration of the TRMT10C–SDR5C1 complex refers to its TRMT10C content. (C and D) Binding data from independent experiments were plotted as single data points against protein concentration and curves fit by non-linear regression. (A and C) (Mt)tRNAIle; (B and D) (mt)tRNAHis.
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gks910-F8: TRMT10C is the subunit primarily responsible for tRNA binding. (A and B) Labelled pre-tRNAs were incubated with increasing concentrations of SDR5C1, TRMT10C or the TRMT10C–SDR5C1 complex and were resolved by native PAGE; representative gels are shown. The concentration of the TRMT10C–SDR5C1 complex refers to its TRMT10C content. (C and D) Binding data from independent experiments were plotted as single data points against protein concentration and curves fit by non-linear regression. (A and C) (Mt)tRNAIle; (B and D) (mt)tRNAHis.

Mentions: We had previously hypothesized that SDR5C1 could contribute to RNA binding in the mtRNase P holoenzyme (17,34) and consequently the methyltransferase subcomplex as well. To investigate whether SDR5C1 is indeed directly involved in the interaction of enzyme and RNA substrate, we compared the tRNA binding properties of the TRMT10C–SDR5C1 complex with those of its individual components. In an electrophoretic mobility shift assay, the TRMT10C–SDR5C1 complex retarded the migration of (mt)pre-tRNAIle and (mt)pre-tRNAHis in a concentration dependent manner (Figure 8A and B, lanes 9–12), and we derived dissociation constants (KD) of 88 ± 8 and 143 ± 21 nM, respectively (Figure 8C and D). Similarly, TRMT10C was binding to both pre-tRNAs, yet, it was causing a smaller shift than the TRMT10C–SDR5C1-tRNA complex, consistent with its lower molecular weight (Figure 8A and B, lanes 5–8). Compared with the TRMT10C–SDR5C1 complex, the pre-tRNA-binding affinity of TRMT10C alone was slightly lower, with an apparent KD of 255 ± 10 nM for (mt)tRNAIle and 203 ± 15 nM for (mt)tRNAHis (Figure 8C and D). SDR5C1 alone, however, did not bind to RNA even at micromolar concentration (Figure 8). These results suggest that SDR5C1 has no direct role in substrate recognition by the TRMT10C–SDR5C1 complex, and TRMT10C is the subunit primarily interacting with the tRNA.Figure 8.


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 is the subunit primarily responsible for tRNA binding. (A and B) Labelled pre-tRNAs were incubated with increasing concentrations of SDR5C1, TRMT10C or the TRMT10C–SDR5C1 complex and were resolved by native PAGE; representative gels are shown. The concentration of the TRMT10C–SDR5C1 complex refers to its TRMT10C content. (C and D) Binding data from independent experiments were plotted as single data points against protein concentration and curves fit by non-linear regression. (A and C) (Mt)tRNAIle; (B and D) (mt)tRNAHis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks910-F8: TRMT10C is the subunit primarily responsible for tRNA binding. (A and B) Labelled pre-tRNAs were incubated with increasing concentrations of SDR5C1, TRMT10C or the TRMT10C–SDR5C1 complex and were resolved by native PAGE; representative gels are shown. The concentration of the TRMT10C–SDR5C1 complex refers to its TRMT10C content. (C and D) Binding data from independent experiments were plotted as single data points against protein concentration and curves fit by non-linear regression. (A and C) (Mt)tRNAIle; (B and D) (mt)tRNAHis.
Mentions: We had previously hypothesized that SDR5C1 could contribute to RNA binding in the mtRNase P holoenzyme (17,34) and consequently the methyltransferase subcomplex as well. To investigate whether SDR5C1 is indeed directly involved in the interaction of enzyme and RNA substrate, we compared the tRNA binding properties of the TRMT10C–SDR5C1 complex with those of its individual components. In an electrophoretic mobility shift assay, the TRMT10C–SDR5C1 complex retarded the migration of (mt)pre-tRNAIle and (mt)pre-tRNAHis in a concentration dependent manner (Figure 8A and B, lanes 9–12), and we derived dissociation constants (KD) of 88 ± 8 and 143 ± 21 nM, respectively (Figure 8C and D). Similarly, TRMT10C was binding to both pre-tRNAs, yet, it was causing a smaller shift than the TRMT10C–SDR5C1-tRNA complex, consistent with its lower molecular weight (Figure 8A and B, lanes 5–8). Compared with the TRMT10C–SDR5C1 complex, the pre-tRNA-binding affinity of TRMT10C alone was slightly lower, with an apparent KD of 255 ± 10 nM for (mt)tRNAIle and 203 ± 15 nM for (mt)tRNAHis (Figure 8C and D). SDR5C1 alone, however, did not bind to RNA even at micromolar concentration (Figure 8). These results suggest that SDR5C1 has no direct role in substrate recognition by the TRMT10C–SDR5C1 complex, and TRMT10C is the subunit primarily interacting with the tRNA.Figure 8.

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.

Show MeSH
Related in: MedlinePlus