MRB10130 predicted protein structure and effect of MRB10130 depletion on T. brucei growth and RNA editing. (A) Amino acid sequence of the MRB10130 ORF. The mitochondrial localization signal was predicted using MitoProt II (Claros and Vincens 1996) and is displayed in bold and underlined. Green ribbons above the sequence represent amino acids predicted for form α-helical structures as modeled using the Phyre2 program (Kelley et al. 2015). (B) Ribbon diagram of the MRB10130 predicted protein structure using Phyre2 intensive protein homology modeling. Each α-helical repeat is displayed using a different color. (C) Repression of MRB10130 by tet-inducible RNAi in PF T. brucei. Cell growth was measured in triplicate for uninduced (tet−) and induced cells (tet+) for 10 d. (D) RNA was isolated from PF T. brucei containing the MRB10130 RNAi construct on day 2 postinduction and quantified by qRT-PCR using primer sets that specifically detect MRB10130 and never-edited, pan-edited, minimally edited, and dicistronic precursor RNAs. Relative RNA abundance represents RNA levels in tet induced cells compared to levels in uninduced cells. RNA levels were normalized to 18S rRNA. Levels and numbers represent the mean and standard error of six determinations. (E) RNA was isolated from PF MRB10130 RNAi cells either grown in the absence (tet−) or presence (tet+) of tet for 2 d, labeled with [α32P]-GTP using guanylyltransferase to identify gRNAs, and resolved on a denaturing gel. Cytoplasmic RNA that was used for normalization and the labeled gRNA are indicated. The level of MRB10130 knockdown was detected for two biological replicates by qRT-PCR (33%–40% of wild-type levels). (F) UV cross-linking assays were performed using radiolabeled gA6[14] gRNA or a 79-nt fragment of pre-edited A6 mRNA with a positive control protein (GST-TbRGG2), a negative control protein (maltose binding protein; MBP), and MBP-MRB10130-His. In vitro transcribed, 32P-labeled RNA was incubated with the indicated purified recombinant protein, and reactions were subjected to UV cross-linking followed by treatment with RNase A. Proteins were resolved by 10% SDS-PAGE. Asterisks indicate the size of full-length proteins (kDa) on both the Coomassie stained gel and the phosphorimage.
