Pentatricopeptide Repeat Proteins Stimulate mRNA Adenylation/Uridylation to Activate Mitochondrial Translation in Trypanosomes

SummaryThe majority of trypanosomal mitochondrial pre-mRNAs undergo massive uridine insertion/deletion editing, which creates open reading frames. Although the pre-editing addition of short 3′ A tails is known to stabilize transcripts during and after the editing, the processing event committing the fully edited mRNAs to translation remained unknown. Here, we show that a heterodimer of pentatricopeptide repeat-containing (PPR) proteins, termed kinetoplast polyadenylation/uridylation factors (KPAFs) 1 and 2, induces the postediting addition of A/U heteropolymers by KPAP1 poly(A) polymerase and RET1 terminal uridyltransferase. Edited transcripts bearing 200- to 300-nucleotide-long A/U tails, but not short A tails, were enriched in translating ribosomal complexes and affinity-purified ribosomal particles. KPAF1 repression led to a selective loss of A/U-tailed mRNAs and concomitant inhibition of protein synthesis. These results establish A/U extensions as the defining cis-elements of translation-competent mRNAs. Furthermore, we demonstrate that A/U-tailed mRNA preferentially interacts with the small ribosomal subunit, whereas edited substrates and complexes bind to the large subunit.

Graphical AbstractFigure optionsDownload high-quality image (187 K)Download as PowerPoint slideHighlights
► PPR proteins KPAF1 and 2 induce postediting addition of 3′ A/U tails by RET1 and KPAP1
► Fully edited mRNAs with 3′ A/U tails associate with translating mitochondrial ribosomes
► Pre-edited mRNAs, gRNAs, and editing complexes bind to large ribosomal subunits
► KPAF1 knockdown ablates A/U tails and inhibits mitochondrial translation in T. brucei