The RNA helicase KREH2 directly binds the RESC1-RESC2 complex in trypanosomatid mitochondrial RNA editing

Trypanosomatids are medically important single-celled eukaryotic protists with exceptional RNA biology. They display highly unusual RNA biogenesis pathways, notably mitochondrial RNA editing, an essential post-transcriptional process transforming cryptic primary transcripts into functional mRNAs. Large multiprotein assemblies coordinate the RNA editing process, in which small guide RNAs (gRNAs) anneal to the pre-mRNAs and serve as templates for uridine insertion or deletion. Kinetoplast RNA Editing Helicases (KREHs) are important components of the RNA editing process that modifies large portions of pre-mRNA via the sequential annealing and dissociation of multiple gRNAs. However, how KREH helicases interact with components of the editing machinery, and which subunits mediate these interactions, remain unresolved. Here, we examined an association between the DEAH/RHA RNA helicase KREH2 and the RESC1-RESC2 protein heterodimer using proximity labeling and co-immunoprecipitation coupled to quantitative mass spectrometry in vivo. We reconstituted a minimal trimeric KREH2 peptide-RESC1-RESC2 complex in vitro and characterized novel direct binding of a KREH2 peptide to RESC1 using cryo-electron microscopy. These results establish a mechanistic link between the RNA helicase KREH2 and the RESC1-RESC2 module at the 5'-end of gRNAs, providing new avenues for studying the orchestration of RNA remodeling in trypanosomatid mitochondrial RNA editing.