Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

We want to thank Kathy Kyler for editing this manuscript, Ken Stuart for supplying monoclonal antisera against RECC subunits, and Laurie K. Read for her gift of polyclonal antisera against GAP1 and RGG2. Funding: National Science Foundation Grant No. NSF1122109 (PI: J.Cruz-Reyes.). NIH/National Institute of Allergies and Infectious Diseases R01 AI088011 (PI: Blaine Mooers). Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103640. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.Yeshttp://www.plosone.org/static/editorial#pee

Targeting RNA Structure to Inhibit Editing in Trypanosomes

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing

The L730V/I RET roof mutations display different activities toward pralsetinib and selpercatinib

Abstract Recently Food and Drug Administration (FDA)-approved pralsetinib (BLU-667) and selpercatinib (LOXO-292) are RET-selective protein tyrosine kinase inhibitors for treating RET-altered cancers, but whether they have distinct activity was unknown. The L730V/I mutations at the roof of the solvent-front site of the RET kinase were identified as strongly resistant to pralsetinib but not to selpercatinib. Selpercatinib effectively inhibited these mutants and the KIF5B-RET(L730V/I) oncogene-driven tumors

RET kinase alterations in targeted cancer therapy

The rearranged during transfection (RET) gene encodes a protein tyrosine kinase. RET alterations by point mutations and gene fusions were found in diverse cancers. RET fusions allow abnormal expression and activation of the oncogenic kinase, whereas only a few of RET point mutations found in human cancers are known oncogenic drivers. Earlier studies of RET-targeted therapy utilized multi-targeted protein tyrosine kinase inhibitors (TKIs) with RET inhibitor activity. These multi-targeted TKIs often led to high-grade adverse events and were subject to resistance caused by the gatekeeper mutations. Recently, two potent and selective RET TKIs, pralsetinib (BLU-667) and selpercatinib (LOXO-292), were developed. High response rates to these selective RET inhibitors across multiple forms of RET alterations in different types of cancers were observed in clinical trials, demonstrating the RET dependence in human cancers harboring these RET lesions. Pralsetinib and selpercatinib were effective in inhibiting RETV804L/M gatekeeper mutants. However, adaptive mutations that cause resistance to pralsetinib or selpercatinib at the solvent front RETG810 residue have been found, pointing to the need for the development of the next-generation of RET TKIs

Photo-crosslinks in REH2 and 3010 pulldowns with an initiating gRNA.

<p>Antibody pulldowns of sedimentation fractions 4-to-6 in mitochondrial extracts from RNAP knockdown cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123441#pone.0123441.g007" target="_blank">Fig 7B</a>) analyzed by (A) Site-specific crosslinking (365-nm UV) with a model initiating gRNA for mRNA A6 that includes a photo-reactive thio-U and ³²P in a single phosphodiester bond, or (B) Western blots of REH2 or GAP1 with size markers in the REH2 IP or the 15S input fractions 4-to-6. (C) Mfold prediction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123441#pone.0123441.ref046" target="_blank">46</a>] of the secondary structure of the gRNA in panel A. The photo-reactive base is circled and guide sequence is inclosed in the gray box. Arrows in A indicate crosslinks in the REH2 or 3010 IP, including at ~30 kDa and, apparently, REH2 itself in the REH2 IP. A mock control (Mk) used an irrelevant affinity-purified antibody.</p

REH2 knockdown affects editing by the initiating gRNA in 3010-MRB but not 3010 association with common MRB1 proteins, gRNA or RECC.

<p>(A) Western blots of REH2 and 3010 in lysates of induced cells with tetracycline (Tet). Full length and proteolytic fragments of REH2 are detected in all Figs in this study. (B) Relative level of early 3’ editing in block 1 of mRNAs A6, ND7, and the first few blocks in RPS12 (RPS). RT-qPCR assays were performed in cell lysates using tubulin as the reference. Uninduced samples are set at 1. (C) 3010-IPs of mitochondrial extracts at 0 or 4 days post-induction in western blots of 3010 and GAP1, or radiolabeled capping (gRNA) and autoadenylation (editing ligases REL1/2). (D) Northern blots of initiating gRNAs for block 1 (B1), gND7 (1269–1319), and gCYb gCYb (54–91) in total mtRNA and 3010-IPs at multiple time points of RNAi induction. The blots were stripped and reprobed for tRNA-Cys, showing that the pulldowns are specific for gRNA.</p

REH2 knockdown affects editing progression.

<p>(A) RT-qPCR of steady-state edited mRNA at block 1 in mRNAs A6 and ND7, and the first few blocks in RPS12 (3’ sites) or a distal block (5’ sites), unedited or never-edted RNAs at days 3, 4, and 5 of REH2 RNAi. Uninduced samples are set at 1.</p