Fig. 4
Divergent effects of KPAF4 knockdown on mitochondrial RNAs. a Northern blotting of pre-edited (Pre-E), partially edited (Part-E), and fully edited RPS12 mRNA variants. Total RNA was separated on a 5% polyacrylamide/8 M urea gel and sequentially hybridized with radiolabeled single-stranded DNA probes. Zero-time point: mock-induced RNAi cell line. Cytosolic 5.8S rRNA was used as loading control. Parent, RNA from parental 29-13 cell line; (dT), RNA was hybridized with 20-mer oligo(dT) and treated with RNase H to show positions of non-adenylated molecules in parental cell line. Pre-edited RNA length increase in KPAF4 RNAi is shown by brackets. Representative of four experiments for edited and three experiments for pre-edited RPS12 mRNA forms are shown. b Alignment of representative RPS12 mRNA 3′ ends in KPAF4 RNAi cells. RNA termini were amplified by cRT-PCR, cloned and sequenced8. A fragment of 3′ untranslated region, short A-tail, and U-extensions are indicated. c Northern blotting of pan-edited A6 mRNA. Total RNA was separated on a 1.7% agarose/formaldehyde gel and sequentially hybridized with oligonucleotide probes for pre-edited and fully edited sequences. Loading control: cytosolic 18S rRNA. Representative of three experiments is shown. d Northern blotting of moderately edited cyb mRNA. Total RNA was separated on a 1.7% agarose/formaldehyde gel and hybridized with oligonucleotide probes for pre-edited and fully edited sequences. Loading control: cytosolic 18S rRNA. Representative of two experiments is shown. e Northern blotting of unedited CO1 and ND1 mRNAs. Total RNA was separated on a 1.7% agarose/formaldehyde gel and sequentially hybridized with oligonucleotide probes. Loading control: cytosolic 18S rRNA. Representative of two experiments is shown. f Northern blotting of mitochondrial ribosomal RNAs. Total RNA was separated on a 5% polyacrylamide/8 M urea gels and hybridized with oligonucleotide probes. Loading control: cytosolic 5.8S rRNA. Representative of two experiments is shown. g Guide RNA northern blotting. Total RNA was separated on a 10% polyacrylamide/8 M urea gel and hybridized with oligonucleotide probes specific for gA6(14) and gCO3(147). Mitochondrially localized tRNACys served as loading control. Single experiment performed
