PRJNA477550
Pentatricopeptide repeat factor KPAF4 functions as poly(A) binding protein in mitochondria of trypanosomes
Most mitochondrial messenger RNAs of trypanosomes undergo U-insertion/deletion editing, and 3'-end adenylation and uridylation. In the causative agent of sleeping sickness, Trypanosoma brucei, internal sequence changes and terminal extensions are tightly coordinated: addition of short (A) tail prior to editing stabilizes edited transcript while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. The enzymes contributing adenosines (KPAP1 poly(A) polymerase) and uridines (RET1 TUTase) have been identified, along with polyadenylation factors required for short A-tail (KPAF3) and long A/U-tail (KPAF1-2) addition. Although participation of a poly(A) binding protein (PABP) in coupling of editing and 3' modification processes has been inferred, the gene encoding a mitochondrial PABP variant appears to be missing from trypanosomal genomes . We report the identification of an essential pentatricopeptide repeat-containing (PPR) Kinetoplast Polyadenylation Factor 4 (KPAF4), which fulfils properties and functions of a poly(A) binding protein. We show that KPAF4 binding to 3' untranslated region and A-tail stabilizes mRNA by impeding 3¬'-5' exonucleolytic activity of the mitochondrial processome. Conversely, KPAF4 binding inhibits RET1 TUTase-catalyzed uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of pre-edited and partially-edited mRNAs. This quality check point ensures translational blockade of mRNAs lacking a complete protein coding sequence. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating a critical interaction between 5'-end bound MERS and 3'-end bound polyadenylation complexes to enable mRNA circularization. This event appears to be critical for transcript stability during the editing process while the post-editing A/U-tailing and translational activation likely require mRNA linearization.