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

Methods Enzymol. 2021-07; 
Inna Aphasizheva, Takuma Suematsu, Andres Vacas, Hong Wang, Chenyu Fan, Xiaojing Zhao, Liye Zhang, Ruslan Aphasizhev
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Catalog Antibody … Elsevier. Methods in Enzymology. Volume 658, 2021, Pages 83-109. Methods in Enzymology … Collect aliquots at all steps to calculate purification fold and yield by quantitative immunoblotting with rabbit polyclonal protein C-tag antibody (GenScript, A00637–40) … Get A Quote

摘要

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 ... More

关键词

Affinity purification, Fluorescent microscopy, Mitochondria, Polyadenylation, Proximity biotinylation, RNA editing, RNA modification, RNA stability, Trypanosoma, UV-crosslinking