Abstract
Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.
Highlights
Trypanosoma brucei and other kinetoplastid protozoa have a single mitochondrion with an unusual mitochondrial genome consisting of many copies of an identical “maxicircle DNA” (~23 kb) and several hundred different types of “minicircle DNAs” (~1 kb)
Our findings suggested that these complexes assemble mRNA-guide RNAs (gRNAs) hybrids and raised the possibility that different MRBs exhibit specialized functions [23]
Our findings indicate that 3010-MRB is relatively enriched with mRNAs that are edited at the first block directed by the initiating gRNA and that REH2 affects multiple editing steps in mRNAs associated with 3010-MRB
Summary
Trypanosoma brucei and other kinetoplastid protozoa have a single mitochondrion with an unusual mitochondrial genome (kDNA) consisting of many copies of an identical “maxicircle DNA” (~23 kb) and several hundred different types of “minicircle DNAs” (~1 kb). RNA editing progresses from 3’ to 5’ along the mRNA through sets of overlapping sequence blocks; the editing of each block is directed by a different gRNA This process exhibits substrate specificity during the life cycle, but the mechanisms involved remain unknown [4]. We have reported two native MRB1 variants (termed here “MRBs”), REH2-MRB and 3010-MRB, with clear differences in protein and RNA composition These MRBs carry unedited and fully edited mRNAs, in addition to gRNA. Finding functionally distinct MRBs that include regulatory proteins and all mRNAs involved in editing, i.e., unedited, partially edited and fully edited transcripts, raises a number of important mechanistic questions that can be directly addressed in the RNA editing of early-branched kinetoplastids
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