Abstract
Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3'-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.
Highlights
Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3-terminal maturation, RNA editing, turnover, and translation; these processes remain poorly studied
REH2 associates, via RNA, with RNA editing core complex (RECC), a battery of accessory editing factors, and mitochondrial ribosomes; thereby, we propose that REH2 ribonucleoprotein complexes (RNPs) are integral components of RNA-linked supramolecular networks that orchestrate the expression of the mitochondrial genome
RNase-resistant REH2-associated proteins that were found in MRB1, TbRGG1, or GRBC1 complexes A, mass spectrometric analyses of proteins were identified in REH2 antibody pulldowns from ϳ20 to 30 S mitochondrial lysate fractions, and unique peptides are shown before or after an extensive nuclease treatment with RNases A, T1, V1, and micrococcal nuclease (MN) while proteins were bound to the beads
Summary
Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3-terminal maturation, RNA editing, turnover, and translation; these processes remain poorly studied. To examine the possibility that REH2 may directly bind RNA, we cross-linked a protein fraction enriched in RECC [3] with the pre-mRNA/gRNA substrate described above but substituted with a photoreactive thio-U and 32P at the editing site [34].
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