The Architecture of Trypanosoma brucei editosomes

Abstract

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra-and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.cross-linking | proteomics | Trypanosoma brucei | RNA editing | editosome T he kinetoplastid Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in subSaharan Africa. Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA, which is composed of interlocked DNA maxicircles and minicircles (1, 2). Several identical maxicircles encode two ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing (3-9). RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).There are three similar versions of ∼20S editosomes that have a common set of 12 proteins (SI Appendix, Fig. S1 and Table S1). However, they are compositionally and functionally distinct in that each...

Surface-driven RNA-refolding by the OB-fold proteins of theTrypanosoma bruceieditosome

“…1c). We also confirmed previous observations [28][29][30] that the different proteins have a high propensity to homo-and hetero-oligomerize using their OB-folds as interaction domains. Interestingly, all identified pair-wise interactions involve TbMP18, the smallest of the editosomal OB-fold proteins, possibly implicating a key role in the assembly of the editosomal complex.…”

“…As a consequence of our hypothesis we postulate that the IDP-domains of the different OB-fold proteins are located on the surface of the 20S editosome. To provide a structural context for the assumption we performed a "coarse-grained" modeling of the structure of the T. brucei editosome using published cryo-EM data 39 and all OB-fold interaction data as spatial restraints [28][29][30] . Interestingly, while the model agrees with the anticipated surface location of the different IDP-domains on the editosome, it also suggests a clustering of all structurally well-defined proteins in the center of the complex.…”

“…Editosome subcomplex reconstitution experiments as well as recent chemical cross-linking experiments demonstrated that the OB-fold proteins of the T. brucei editosome interact with each other [28][29][30] . This tempted us to analyze the association behavior of the different protein preparations in "mix-and-match-type" binding experiments.…”

“…For that we used the cryo-EM structure of the T. brucei 20S editosome 39 . As spatial restraints we implemented all above-described OB-fold interaction data as well as all published binary interactions of editosome components including the recent chemical cross-linking data of the T. brucei OB-fold proteins [28][29][30] . Structure coordinates for the individual proteins were taken from Czerwoniec et al, 2015 17 (and references therein) and all disordered regions were simulated as coarse-grained flexible shapes.…”

“…The data suggest a scenario in which TbMP18, TbMP24, TbMP42, TbMP63 and TbMP81 interact with each other, mainly relying on their OB-fold domains thereby forming a clustered "OB-fold core" within the 20S editosome 29 . Recent interprotein chemical cross-linking data support the existence of such a "core-domain" and have identified cross-links between all six OB-fold proteins most of which within or in proximity to the OB-fold domains 30 . Importantly, clustered OB-folds in the yeast RRP44 protein have been demonstrated to catalyze the unwinding of dsRNA 31,32 .…”