Multiple domains of the integral KREPA3 protein are critical for the structure and precise functions of RNA Editing Catalytic Complexes inTrypanosoma brucei

Abstract

The gRNA directed U-insertion and deletion editing of mitochondrial mRNAs that is essential in different life cycle stages for the protozoan parasite Trypanosoma brucei is performed by three similar multi-protein catalytic complexes (CCs) that contain the requisite enzymes. These CCs also contain a common set of eight proteins that have no apparent direct catalytic function, including six that have an OB-fold domain. We show here that one of these OB-fold proteins, KREPA3 (A3), has structural homology to other editing proteins, is essential for editing and is multifunctional. We investigated A3 function by analyzing the effects of single amino acid loss of function mutations most of which were identified by screening bloodstream form (BF) parasites for loss of growth following random mutagenesis. Mutations in the ZFs, an intrinsically disordered region (IDR) and several within or near the C-terminal OB-fold domain variably impacted CC structural integrity and editing. Some mutations resulted in almost complete loss of CCs and its proteins and editing whereas others retained CCs but had aberrant editing. All but a mutation which is near the OB-fold affected growth and editing in BF but not procyclic form (PF) parasites. These data indicate that multiple positions within A3 have essential functions that contribute to the structural integrity of CCs, the precision of editing and the developmental differences in editing between BF and PF stages.

Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4

“…Several RECC protein mutagenesis studies, including of B5-B8 that contain RNase III-like domains, have identified single amino acid substitutions with different consequences on BF vs. PF cell growth and RECC integrities (Carnes et al, 2011;McDermott et al, 2015a, b;McDermott and Stuart, 2017;McDermott et al, 2019;Carnes et al, 2022;Davidge et al, 2023). Therefore, we hypothesized that we might also observe life cycle stage differences in the growth of cells containing substitutions that were identified in our BF B4 deep mutational scan.…”

“…Functional annotation of evolutionarily divergent trypanosomatid proteins with limited sequence homology to other eukaryotic proteins can be advanced by mutagenesis combined with novel high-throughput functional analyses (McDermott et al, 2015a;Carnes et al, 2022;Davidge et al, 2023). Here we describe the development of a deep mutational scanning approach for T. brucei, which makes use of tet-conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants (McDermott et al, 2015a;McDermott and Stuart, 2017), a high-throughput sequencing readout for cell growth upon exclusive expression of mutant variants (Wei and Li, 2023), and freely available computational tools to estimate variant fitness (Faure et al, 2020).…”

“…Here we describe the development of a deep mutational scanning approach for T. brucei, which makes use of tet-conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants (McDermott et al, 2015a;McDermott and Stuart, 2017), a high-throughput sequencing readout for cell growth upon exclusive expression of mutant variants (Wei and Li, 2023), and freely available computational tools to estimate variant fitness (Faure et al, 2020). The method has improved upon our previous random mutagenesis and complementation approach (McDermott et al, 2015a;Carnes et al, 2022;Davidge et al, 2023) by increasing the number of substitutions that can be phenotypically screened by two orders of magnitude. This was enabled by both the high-throughput sequencing readout, and the generation of larger variant libraries in BF T. brucei via TMP/ddDHFR-induced I-SceI meganuclease stabilization and cleavage at the mutant library target locus.…”

“…However, functional studies of many proteins in T. brucei and related parasites have been hampered by limited sequence homology to proteins from other model organisms, which inhibits our ability to identify the amino acids most critical for function. We previously described mutagenesis and complementation screens that we used to measure the effects of different randomly generated substitutions on T. brucei cell fitness (McDermott et al, 2015a;Carnes et al, 2022;Davidge et al, 2023). However, these earlier screens restricted analysis to a relatively small number of substitutions due to low stable cell transfection efficiencies, in addition to complex and timeconsuming cell handling procedures.…”

“…Finally, we show that several substitutions identified by our screen as detrimental to BF cell fitness do not affect procyclic form (PF) cell growth or RECCs. Thus, the B4 RNase III-like domain is also involved in the differences in RECCs that occur between BF and PF life cycle stages (McDermott et al, 2015a(McDermott et al, , b, 2019Carnes et al, 2022;Davidge et al, 2023).…”