Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing

Abstract

In Trypanosoma brucei , the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)–programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo–electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA–binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome’s catalytic modality.

KREH2 helicase represses ND7 mRNA editing in procyclic-stageTrypanosoma bruceiby opposite modulation of canonical and “moonlighting” gRNA utilization creating a proposed mRNA structure

“…All three core proteins in REH2C co-purified with mRNA substrates and products (fully and partially edited) in the absence of RESC, i.e., upon depletion of RESC1 and gRNA (32). Conversely, several purifications of RESC proteins have detected KREH2 (27–30), supporting the speculation that KREH2 could mediate remodeling between RESC-A and RESC-B CryoEM reconstructions (36). KREH2 immunoprecipitated from mt-extract or recombinantly expressed, crosslinked with RNA, and supported dsRNA unwinding in vitro (41,42).…”

“…We compared total mtRNA in PCF and BSF and RNA-immunoprecipitations (RIPs) of RESC6 and RESC1 in PCF. Among many potential RESC isoforms (30,31,63), RESC-A contains RESC6 and RESC1, while RESC-B contains RESC6 but not RESC-1 (36). This is the first time isolated native RESC1 complexes have been examined by RNA-Seq, as was previously done for native RESC6 complexes (13,38).…”

“…We and others initially showed that purified RESC is enriched with editing substrates and products (31,33). Two RESC cryoEM reconstructed structures (RESC-A and RESC-B) may represent gRNA storage and mRNA-gRNA duplex substrate complexes, respectively (36,37). R NA E diting Helicase KRE H2 -associated C omplex (REH2C) (4,5,15) contains three core proteins: the typifying DEAH-Box RNA helicase KREH2 and two directly bound helicase factors, the zinc finger protein KH2F1 and KH2F2.…”

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment

“…Swiss-Model ( 72 ) was utilized to model complete B. nonstop RESC-A and RESC-B complexes and their individual subunits using T. brucei cryo-electron microscopy (cryo-EM) structures ( 26 ) as templates. To ensure that homology models were not biased towards the templates in localized regions, RESC-A complex and its subunits were also modelled with AlphaFold v. 2.3.0 ( 73 ) using a sequence database customized for trypanosomatids ( 74 ).…”

“…The complexity of RNA editing is also apparent in the 60+ proteins that either in complex or solo are part of the enzymatic, structural, and regulatory machinery necessary to execute these modifications ( 19 , 26 ). The RNA editing catalytic complex (RECC) and RNA editing substrate-binding complex (RESC) that comprise the bulk of the editing machinery are now much better understood than the genetic role of editing itself ( 17 , 27–29 ).…”