FIGURE 5.

Mne1 contributes to the splicing of the aI5β intron. A, RT-PCR analysis of aI5β splicing defect in mne1Δ cells with and without mitochondrial introns. The schematic depicts primers used for RT-PCR. The upper panel shows COX1 mRNA with (unspliced) and without (spliced) aI5β intron. The bottom panel shows amplification of intronless COX2 mRNA that served as a loading control. The PCR products were amplified with 16 cycle reactions. B, RNase protection assay for aI5β splicing defect. Mitochondrial mRNAs were isolated from WT, mrs1Δ, pet54Δ, mss18Δ, and mne1Δ cells. Schematics show the identity of the protected fragments, COX1 pre-mRNA containing aI5β intron and COX1 ligated exon. The asterisk depicts incomplete digestion of the single-stranded portion of the pre-mRNA bound probe. M1 and M2, markers; probe, no RNase control; tRNA, no mitochondrial RNA control. The inset shows rRNA levels that were used as a loading control.