Fig. 3: Mitochondrial SAM is critical for processing of the rRNA gene cluster.

a ONT sequencing data filtered for all reads containing tRNA sequences and mapped against the mitochondrial genome (KR020497). Control (Ctrl; green) and Samc KO (red) from 8 and 12-week-old muscle (quad) and MEF samples (n = 3 biologically independent samples). b ONT sequencing data filtered for reads containing tRNA sequences present in more than 0,01% of the total RNA pool and mapped against the mitochondrial genome (KR020497). Control (inner circle; green) and Samc KO (outer circle; pink) reads are shown from 8 and 12-week-old quadriceps and MEF samples. Black dots represent presence of a tRNA sequence at the 5′ or 3′ termini of the reads. Shown are the fractions of transcripts passing through at least the centre of one tRNA, calculated relative to reads in the next mRNA or rRNA. (n = 3 biologically independent samples). c–e Zoom-in of KO^MEFs RNA reads passing through (b) 12S rRNA, (c) 16S rRNA, or (d) mtNd1 with no fraction cut-off. Gene borders are indicated by dotted lines. (n = 3 biologically independent samples). f Northern blot analysis of rRNA mitochondrial transcripts and flanking tRNAs in control and Samc KO MEFs as indicated (n = 5 biologically independent samples). Nucleotide length provided in grey (nts). Black arrowheads indicate unprocessed transcripts (1) mtF-12S-mtV−16S-mtL1-mtNd1 (2) mtF-12S-mtV-16S, (3) mtV-16S. g number and position of reads containing mtF (tRNA^Phe) in KO^MEFs is shown. Transcription start sites for the heavy strand promoter are indicated in bold. (n = 3 biologically independent samples). Number of reads and percentage of total reads is indicated. h Northern blot analysis of selected mitochondrial transcripts in control or Samc KO MEFs after treatment with 20 mM IMT1B, a mitochondrial transcription inhibitor, using probes against mtF (top panel), mtV (middle panel) or 12S (bottom panel). Nucleotide length provided in grey (nts). (*indicates tRNAs for which the gel resolution does not allow us to distinguish whether they are present or absent.) 18S was used as loading control. Treatment times were 8 and 16 h, chase timepoints were at 4, 8, 12 and 24 hours. 0 = dimethyl sulfoxide (DMSO) vehicle control without IMT1B). Representative experiment of three independent experiments. Source data are provided as a Source Data file.