SUMMARY Small non-coding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5′ triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3′ processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3′-5′ exonuclease and three additional subunits. This complex, termed mitochondrial 3′ processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature size of 40–60 nucleotides, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3′-5′ degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5′ extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3′-5′ exonucleolytic degradation.

Conventional tRNAs have highly conserved sequences, four-armed cloverleaf secondary structures, and L-shaped tertiary structures. However, metazoan mitochondrial tRNAs contain several exceptional structures. Almost all tRNAsSer for AGY/N codons lack the D-arm. Furthermore, in some nematodes, no four-armed cloverleaf-type tRNAs are present: two tRNAsSer without the D-arm and 20 tRNAs without the T-arm are found. Previously, we showed that in nematode mitochondria, an extra elongation factor Tu (EF-Tu) has evolved to support interaction with tRNAs lacking the T-arm, which interact with C-terminal domain 3 in conventional EF-Tu. Recent mitochondrial genome analyses have suggested that in metazoan lineages other than nematodes, tRNAs without the T-arm are present. Furthermore, even more simplified tRNAs are predicted in some lineages. In this review, we discuss mitochondrial tRNAs with divergent structures, as well as protein factors, including EF-Tu, that support the function of truncated metazoan mitochondrial tRNAs.

In , most mitochondrial mRNAs undergo internal changes by RNA editing and 3' end modifications. The temporally separated and functionally distinct modifications are manifested by adenylation prior to editing, and by post-editing extension of a short A-tail into a long A/U-heteropolymer. The A-tail stabilizes partially and fully edited mRNAs, while the A/U-tail enables mRNA binding to the ribosome. Here, we identify an essential pentatricopeptide repeat-containing RNA binding protein, kinetoplast polyadenylation factor 3 (KPAF3), and demonstrate its role in protecting pre-mRNA against degradation by the processome. We show that KPAF3 recruits KPAP1 poly(A) polymerase to the 3' terminus, thus leading to pre-mRNA stabilization, or decay depending on the occurrence and extent of editing., KPAF3 stimulates KPAP1 activity and inhibits mRNA uridylation by RET1 TUTase. Our findings indicate that KPAF3 selectively directs pre-mRNA toward adenylation rather than uridylation, which is a default post-trimming modification characteristic of ribosomal and guide RNAs. As a quality control mechanism, KPAF3 binding ensures that mRNAs entering the editing pathway are adenylated and, therefore, competent for post-editing A/U-tailing and translational activation.

SummaryTranslation elongation factor G (EF-G) in bacteria plays two distinct roles in different phases of the translation system. EF-G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post-termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF-G are distributed over two different EF-G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF-G is also found in bacteria. Two EF-G paralogues are found in the spirochaete Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-G1 is a translocase, while EF-G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF-G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF-3) is present in the reaction. These results indicate that two B. burgdorferi EF-G paralogues are close relatives to mitochondrial EF-G paralogues rather than the conventional bacterial EF-G, in both their phylogenetic and biochemical features.

Mitochondrial genomes are often transcribed into polycistronic RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that in Trypanosoma brucei the monophosphorylated 5′ ends of functional molecules typify precursor partitioning by an unknown endonuclease. On the contrary, we demonstrate that individual mRNAs and rRNAs are independently synthesized as 3′-extended precursors. The transcription-defined 5′ terminus is converted into a monophosphorylated state by the pyrophosphohydrolase complex, termed the “PPsome.” Composed of the MERS1 NUDIX enzyme, the MERS2 pentatricopeptide repeat RNA-binding subunit, and MERS3 polypeptide, the PPsome binds to specific sequences near mRNA 5′ termini. Most guide RNAs lack PPsome-recognition sites and remain triphosphorylated. The RNA-editing substrate-binding complex stimulates MERS1 pyrophosphohydrolase activity and enables an interaction between the PPsome and the polyadenylation machinery. We provide evidence that both 5′ pyrophosphate removal and 3′ adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3′–5′ exonucleolytic trimming defines the mRNA 3′ end before adenylation. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.