CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei

Methods Enzymol. 2021;658:83-109. doi: 10.1016/bs.mie.2021.06.004. Epub 2021 Jul 14.

Abstract

Unicellular parasite Trypanosoma brucei maintains an elaborate mitochondrial mRNA processing pathway including 3'-5' exonucleolytic trimming of primary precursors, 5' and 3' modifications, and, in most cases, massive U-insertion/deletion editing. Whereas the role of editing in restoring protein coding sequence is apparent, recent developments suggest that terminal modifications are equally critical for generating a stable translationally competent messenger. The enzymatic activities responsible for 5' pyrophosphate hydrolysis, 3' adenylation and uridylation, and 3'-5' decay are positively and negatively regulated by pentatricopeptide repeat-containing (PPR) proteins. These sequence-specific RNA binding factors typically contain arrays of 35-amino acid repeats each of which recognizes a single nucleotide. Here, we introduce a combinatorial CTS affinity tag, which underlies a suite of methods for PPR proteins purification, in vivo RNA binding sites mapping and sub-cellular localization studies. These approaches should be applicable to most trypanosomal RNA binding proteins.

Keywords: Affinity purification; Fluorescent microscopy; Mitochondria; Polyadenylation; Proximity biotinylation; RNA editing; RNA modification; RNA stability; Trypanosoma; UV-crosslinking.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Mitochondria / genetics
  • Mitochondria / metabolism
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • RNA Editing
  • RNA, Mitochondrial / genetics
  • RNA, Mitochondrial / metabolism
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism
  • Trypanosoma brucei brucei* / genetics
  • Trypanosoma brucei brucei* / metabolism

Substances

  • Protozoan Proteins
  • RNA, Mitochondrial
  • RNA, Protozoan