Figure 2.

Generation of Mettl20 knockout mouse. (a) Schematics of mouse Mettl20 gene. Mettl20 has five transcripts. Transcript 1 to 4 encode same longer isoform (a) but possess different 5′ UTR, whereas transcript 5 encodes shorter isoform (b), which start translation in exon4. A guide RNA was designed against the Ex4. Small box; transcript including UTRs, large box; coding region, filled box; MTase domain, arrow head; gRNA target site. (b) DNA sequence of the Mettl20 KO mice. The KO mice lost 17 base pairs around the gRNA target site which resulted in a frame-shift mutation of Mettl20. (c) Protein expression of METTL20 in WT and KO mice. Several tissues were collected from WT and KO mice, and protein expression of mMETTL20 or ETFB were measured with a western blotting with anti-Mettl20 antibodies (top) or anti-ETFB antibodies (bottom). (d) Methylation of endogenous ETFB in the mice liver. Mitochondria were fractionated from WT or Mettl20 KO mice liver, and endogenous ETFB were immunoprecipitated with anti-ETFB antibodies. Precipitated proteins were separated with SDS-PAGE, and their methylation were detected with anti-triMeLys antibodies (top), anti-diMeLys antibodies (mixture of c4930 and c5123) (middle), and anti-ETFB antibodies (bottom, as loading control). (e) nanoLC-MS analysis of endogenous ETFB. Immunoprecipitated ETFB were separated with SDS-PAGE, digested with Asp-N, and methylation of peptides containing K200/203 were analyzed with nanoLC-MS (Q-Exactive, Thermo Fisher Scientific). Detected triply charged peptide ion were m/z = 904.869 (z = 3), which corresponds to un-modified EPRYATLPNIMKAKKKKIEVVKAG peptide in KO mice and m/z = 932.900 (z = 3), which corresponds to the peptide carrying 6 methyl groups in WT.