ИСТИНА

Ribosomal RNA methylation occurs in all organisms and such modification may influence ribosome assembly, structure and function. Recently the mapping of all methylated rRNA nucleotides in model organisms has been completed and all proteins responsible for such modifications in E. coli and S. cerevisiae have been discovered. However, the identification of many eukaryotic rRNA methyltransferases and functional studies of corresponding modified nucleotides still need to be carried out. E. coli 16S rRNA contains a unique m4C modification, for formation of which enzyme rsmH is known to be responsible. Mammalian cytoplasmic rRNAs lack this type of methylation, but there is one m4C nucleotide in mitochondrial 12S rRNA. Mammalian mitochondrial protein METTL15 is a homolog of rsmH, which makes it the candidate for 12S rRNA m4C methyltransferase. Following the similar logic, we suggested that m5U in 12S rRNA may be formed by TRMT2B. To test our hypotheses, we used CRISPR/Cas9 to generate two KO cell lines from murine NS0 cells by introducing a frameshift mutation to TRMT2B or METTL15 ORF. Using biotinylated oligonucleotides, we isolated 12S rRNA fragments containing the positions of studied modifications from WT and KO cell lines and analyzed them by MALDI MS. Observed mass spectra proved the lack of methylation in KO cell lines. In order to eliminate the possibility of off-target effects impacting our results, we are planning to integrate functional ORFs of the proteins into genomes of KO cell lines to restore 12S rRNA modification. Also, we are going to perform a comparison of KO and WT proteomes. As we suggest that the absence of these methylations may influence translation efficiency in mitochondria, we are planning to investigate respiratory chain activity in KO and WT cell lines. These results may shed light on functional role of mitochondrial rRNA methylation.