Figure 1.

Guide RNA Biogenesis Model. Bidirectional transcription of gRNA gene generates sense and antisense precursors with overlapping 5′ regions. Mitochondrial 3′ processome-embedded RET1 TUTase and DSS1 3′–5′ exonuclease catalyze 3 coupled reactions: primary precursor uridylation, processive precursor degradation and secondary uridylation of trimmed gRNA. Primary uridylation stimulates DSS1hydrolytic activity, which provides energy for unwinding the secondary structures along gRNA precursor. The MPsome stochastically pauses at 10–12 nt from stable duplex regions formed by head-to-head hybridization of sense and antisense primary transcripts. The MPsome pausing allows RET1 TUTase to engage the single-stranded 3′ overhang and perform secondary uridylation. This step may disengage the MPsome from the duplex intermediate. Double-stranded RNA intermediate likely undergoes unwinding before mature gRNA can be sequestered by the gRNA binding complex and delivered into the editing pathway.