Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid

Abstract

Background: Mitochondrial rRNAs contain site-specific modifications, including methylated residues, but few of the enzymes responsible for these modifications have been found. Results: RNMTL1, MRM1, and MRM2 are methyltransferase family members found associated with the mtDNA nucleoid and with the large ribosomal subunit. Conclusion:The association of putative RNA-modifying enzymes with nucleoids and ribosomes supports the model that assembly of mitochondrial ribosomes begins while rRNA transcription continues. Significance: This work advances understanding of the assembly of mitochondrial ribosomes necessary for biosynthesis of the respiratory chain.

Assignment of 2′-O-Methyltransferases to Modification Sites on the Mammalian Mitochondrial Large Subunit 16 S Ribosomal RNA (rRNA)

“…The large subunit 16 S rRNA modifications include three 2Ј-O-ribose methylations at G 1145 , U 1369 , and G 1370 and a pseudouridylation at U 1397 . We have identified RNMTL1 as a novel mitochondrial rRNA methyltransferase localized at nucleoids involved in the 2Ј-O-methylation of G 1370 , a unique site that is not methylated in bacterial or yeast mitochondrial rRNA (38). Studies in yeast mitochondria suggest that human homologs of MRM1 and MRM2, both of which we found to co-localize with nucleoids, are involved in 16 S rRNA methylation.…”

“…Silencer Select siRNA (Ambion) sequences are as follows: siMRM1-A (5Ј-CCAGGCCCAUGUUUAUUGA-3Ј), siMRM1-B (5Ј-GGAAGUUGAGGGAAAGUUU-3Ј), siMRM2-A (5Ј-ACA-UCUCAGGGACCCAUUU-3Ј), and siMRM2-B (5Ј-GAAUGU-AAGGAUCAUCAAA-3Ј). HeLa cells were reverse-transfected with 3 or 6 nM siRNA and Lipofectamine RNAiMAX (Life Technologies) for 3 days, as described (38). Antibodies are listed with their suppliers and dilutions: MRM1 (Sigma-Aldrich, 1:3,000), MRM2 (Abcam; 1:1,000), and succinate dehydrogenase subunit A (SDHA, MitoSciences, 1:10,000).…”

“…The biotinylated probe was generated by in vitro T7 RNA polymerase transcription of a sequence complementary to residues 142-458 of 16 S rRNA that was cloned into pBS Ϫ plasmid in the presence of biotinylated CTP. In vitro-synthesized full-length 16 S RNA was transcribed from a pBSKII ϩ plasmid with a 16 S RNA insert as described (38).…”

“…Assignment of Mitochondrial Methyltransferases to Target Sites-We have previously used siRNA to significantly reduce the levels of RNMTL1 protein in HeLa cells (38). In this analysis, primer extension in the presence of limiting deoxynucleotide triphosphate concentrations was used to conclude that RNMTL1 methylates residue G 1370 of 16 S rRNA, but this analysis was complicated somewhat by methylation of the adjacent U 1369 residue.…”

“…One of the most common methods involves inhibition of reverse transcriptase at low deoxynucleotide triphosphate levels (47), as we have recently applied (38). However, a method that has not received much attention is the use of DNAzymes, deoxyoligonucleotides that can anneal to RNA and direct cleavage at a specific unmodified site.…”