MRB7260 is essential for productive protein-RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing

Abstract

The trypanosome RNA editing substrate binding complex (RESC) acts as the platform for mitochondrial uridine insertion/deletion RNA editing and facilitates the protein-protein and protein-RNA interactions required for the editing process. RESC is broadly comprised of two subcomplexes: GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex). Here, we characterize the function and position in RESC organization of a previously unstudied RESC protein, MRB7260. We show that MRB7260 forms numerous RESC-related complexes, including a novel, small complex with the guide RNA binding protein, GAP1, which is a canonical GRBC component, and REMC components MRB8170 and TbRGG2. RNA immunoprecipitations in MRB7260-depleted cells show that MRB7260 is critical for normal RNA trafficking between REMC and GRBC. Analysis of protein-protein interactions also reveals an important role for MRB7260 in promoting stable association of the two subcomplexes. High-throughput sequencing analysis of RPS12 mRNAs from MRB7260 replete and depleted cells demonstrates that MRB7260 is critical for gRNA exchange and early gRNA utilization, with the exception of the initiating gRNA. Together, these data demonstrate that MRB7260 is essential for productive protein-RNA interactions with RESC during RNA editing.

Keywords: RNA editing substrate binding complex; Trypanosoma brucei; guide RNA; guide RNA binding complex; kinetoplastida; uridine insertion/deletion RNA editing.

FIGURE 1.

MRB7260 subcellular localization and effect of MRB7260 depletion on T. brucei growth and RNA editing. (A) PF parental 29-13 cells or MRB7260-MHT cells were used to determine the subcellular localization of MRB7260 by indirect immunofluorescence (Myc; green). Mitochondria were detected using MitoTracker Red CMXRos (Mito; red). DAPI was used to stain nuclei and kinetoplasts (blue). Signals are merged on the right (DAPI/Myc and Mito/Myc). (DIC) Differential interference contrast. (B–E) MRB7260 was repressed by tet-inducible RNAi in PF (B,C) and BF (D,E) T. brucei, and cell growth was measured in triplicate for uninduced (tet−) and induced cells (tet+) for 10 d (B,D). RNA was isolated from procyclic (C) and bloodstream (E) form T. brucei on day 3 post-induction. RNA was quantified by qRT-PCR using primer sets that specifically detect 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. (F) RNA was isolated from PF MRB7260 RNAi cells either grown in the absence (tet−) or presence (tet+) of tet for 3 d and then labeled with [α³²P]-GTP using guanylyltransferase to identify gRNAs and resolved on a denaturing gel. A cytoplasmic RNA that was used for normalization and the labeled gRNA are indicated. Biological replicate experiments were performed for B–F, and the level of MRB7260 knockdown was validated using qRT-PCR (PF: 26%–40% remaining, BF: 50% remaining). See also Materials and Methods and Supplemental Figure S6 for an estimate of remaining protein.

FIGURE 2.

Analysis of RPS12 transcripts from MRB7260 replete and depleted cells at the sequence level using TREAT. (A) The average number of normalized pre-edited RPS12 transcripts for the 10 uninduced samples (Avg Un), and two MRB7260 RNAi-induced samples (7260) in PF T. brucei. (B) RPS12 edited mRNA sequence with the exacerbated pause sites (EPSs) that arise upon depletion of MRB7260 for both biological replicates indicated with black diamonds. Solid black diamonds represent MRB7260 EPSs within the gRNA-directed region, whereas black diamonds with red outlines indicate MRB7620 EPSs near gRNA ends. Blue diamonds show EPS from a previous study (Simpson et al. 2017) for the knockdown of TbRGG2. Gray bars below the sequences display regions guided by specific gRNAs as reported previously (Koslowsky et al. 2014) and are numbered from the 3′ to 5′ direction along the mRNA. gRNA anchor regions at the 5′ of the gRNA are depicted with bold lines, whereas the hatched regions at the 5′ and 3′ ends represent variation in gRNA lengths within that class of gRNA. The start and stop codons of the coding sequence are underlined. Open circles indicate editing stop sites in which junction length zero is significantly increased when MRB7260 is depleted in both replicates compared to the average of the uninduced samples. Underscores are shown for clarity in stretches of unedited sequence so that numbers above align with the correct ES. (C) Average number of RPS12 transcripts for the 10 uninduced (Avg Un) and the two MRB7260 RNAi-induced (7260) samples at each EPS for the MRB7260 knockdown.

FIGURE 3.

Sequence analysis of the most abundant junctions that occur within the second gRNA block MRB7260 replete and depleted cells. Average number (normalized counts) of junction sequences in the uninduced samples (Unin) and the MRB7260 knockdown (KD) from editing stop sites within the second gRNA guided block (editing stop sites 22–40). ESS refers to the editing stop site for that sequence, whereas Jcn Len indicates the number of editing sites that the junction sequence spans. Fold indicates the fold change in the number of sequences for each specific junction in the MRB7260 RNAi samples compared to the uninduced samples. Pre-edited sequences are displayed in black, edited RNA is indicated in red, while junctions are shown in green. Blue lines below the pre-edited and edited transcripts show the gRNA-directed blocks. The positions of the ES are indicated with triangles and are numbered from 3′ to 5′. EPSs that arise following MRB7260 depletion are shown with black diamonds, with sites within the gRNA-directed blocks (all black) or near gRNA ends (black with red outline) as in Figure 2. The most abundant junction sequences within the gRNA-2 directed block are listed and grouped according to those that indicate a defect in editing progression (A) or correct gRNA exchange (B) when MRB7260 is depleted.

FIGURE 4.

MRB7260 protein–protein interactions. (A) PF T. brucei cells harboring the MRB7260 RNAi construct were grown for 3 d in the presence or absence of tet. Cell lysates were then analyzed by western blot using antibodies for components of the GRBC subcomplex, the REMC subcomplex, and RECC. Western blot using an antibody against p22 served as a load control. MRB7260 knockdown was detected by qRT-PCR (∼20% of wild type levels). Figure shown is representative of two technical replicates. (B) Schematic representation of the RESC complex divided into the GRBC and REMC subcomplexes. Solid lines (strong) or dotted lines (weak) indicate the strength of interaction observed in a published yeast two-hybrid screen (Ammerman et al. 2012) and with MRB7260 performed in this study. Interactions observed in both directions are shown using thick lines, whereas thin lines represent interactions that occurred in one direction of the screen. These direct protein–protein interactions were verified using immunoprecipitation (IP) to place each protein into either the REMC or GRBC subcomplexes (Ammerman et al. 2012, 2013). (C) A subset of the direct protein interactions tested using a yeast two-hybrid screen. pGAD-T7 is a vector used to express prey activation domain fusion proteins, while pGBK-T7 expressed the bait binding domain fusion proteins. Yeast cells were grown on media lacking leu and trp (-L-W) to select for cotransformants and then subsequently grown on plates lacking leu, trp, and his but supplemented with 1 mM 3-AT to select for bait–prey interactions. Interactions were then scored according to previously described strong (MRB3010-MRB8620, +++++) or weak (MRB11870-GAP1, +++) interactions (Ammerman et al. 2012). Results are representative of at least two biological replicates for each interaction. (D) MRB7260-MHT (Myc-His-TAP) and associated proteins were affinity purified from PF T. brucei cell extracts that were either RNase inhibited (RNA+) or RNase treated (RNA−). Proteins were eluted from IgG beads using TEV protease cleavage and electrophoresed on 10% (w/v) SDS-PAGE gels, followed by western blotting using antibodies against myc (to detect MRB7260) and various mitochondrial proteins. For visualization, 0.025% of input and 1% of TEV elutions were loaded. Arrows indicate the change in protein association with MRB7260 when RNA is removed. Representative of two biological replicate experiments.

FIGURE 5.

Visualization of MR7260 complexes using gradient sedimentation. (A, top) Stepwise description of the method used to visualize the MRB7260-containing complexes. (Bottom) MRB7260-MHT was affinity purified from PF T. brucei in the presence or absence of RNA and then subjected to 10%–30% (v/v) glycerol-gradient sedimentation. Fractions collected were analyzed by western blot with antibodies against RESC proteins. Size markers obtained from a parallel gradient are displayed above the fraction numbers. Representative western blots of two biological replicate experiments. Schematic representation of the proteins found in the fractions is displayed below the blots. Dotted borders around the proteins indicate a decreased presence of the protein in a given fraction when RNA is removed. Gray circles indicate unknown proteins that may also be present within these complexes. Pictures show the presence or absence of proteins and are not meant to indicate stoichiometry. (B) Total cell lysate (not treated with RNase) from the parental PF T. brucei strain 29-13 cell line was subject to 10%–30% (v/v) glycerol gradient sedimentation. Fractions were collected and analyzed by western blot with antibodies against MRB7260. Shown is a representative of two biological replicate experiments.

FIGURE 6.

Effect of MRB7260 depletion on RESC protein–protein interactions. (A) PF T. brucei cells harboring both the MRB7260 RNAi construct and the TbRGG2-HTM construct were used to assess the proteins associated with the REMC subcomplex in the absence of MRB7260. Cells were grown for 3 d in the absence or presence of tet, followed by IP of TbRGG2 using the myc tag. Bound protein was eluted from myc beads using TEV protease cleavage. Elutions were then analyzed using western blot with a subset of REMC and GRBC protein antibodies. Western blots display 0.025% of input and 6% of TEV elution. Biological replicate experiments were performed and qRT-PCR was used to validate the level of MRB7260 knockdown (29%–44% remaining). (B) Quantification of western blots in A using BioRad Image Lab software. Protein levels were normalized to amount of TbRGG2 for that immunoprecipitation (IP). The protein associated with TbRGG2 was then calculated by comparing the normalized protein levels from the tet+ IP to that of the tet− IP (which was set to 100%). Bar graphs represent the average and standard deviation of two biological replicates with a total of n = 4 for each antibody. (C) PF T. brucei cells containing both the MRB7260 RNAi construct and the MRB3010-PTP construct were used to identify proteins associated with GRBC when the levels of MRB7260 are altered. Cells were either induced with tet or left uninduced for 3 d followed by IP of MRB3010 using IgG. Protein elutions that resulted from TEV protease cleavage were then analyzed using western blot, with 0.015% of input and 1% of TEV elution loaded. Biological replicate experiments were performed and qRT-PCR was used to validate the level of MRB7260 knockdown (50%). (D) Quantification of western blots in C using BioRad Image Lab software. Protein levels were normalized to the amount of MRB3010 for that IP. The protein associated with MRB3010 was then calculated by comparing the normalized protein levels from the tet+ IP to that of the tet− IP (which was set to 100%). Bar graphs represent the average and standard deviation of two biological replicates with a total n = 4 for each antibody.

FIGURE 7.

Effect of MRB7260 depletion on RESC protein–RNA interactions. (A) Schematic representation of the qRT-PCR primers used to detect the largest pool of mRNAs for a given transcript in RIPs. Pre-edited regions are indicated in gray and edited RNA is displayed using hashed lines. RIP mRNA primers hybridize to the 5′ never-edited (indicated in black) and 5′-most pre-edited region of the transcripts. RIP primers detect pre-edited and all partially edited RNAs, excluding RNAs that are fully edited. (B) Total cellular RNA from PF cells containing the MRB7260 RNAi construct grown with or without tet for 3 d were analyzed using the RIP qRT-PCR primers. Relative RNA abundance represents RNA levels in tet-induced cells compared to uninduced. RNA levels were normalized to 18S rRNA levels. For comparison, results with primers that detect only fully edited A6 mRNA (as in Fig. 1) are also displayed. qRT-PCR was also used to validate the level of MRB7260 knockdown in the biological replicate experiments (32%–48%). (C,E) Comparison of RNA immunoprecipitated with TbRGG2-HTM (C) or MRB3010-PTP (E) compared to a mock IP. RNA was detected using RIP mRNA primers described in A and primers designed to detect a subset of gRNAs using qRT-PCR. RNA levels were standardized against 18S rRNA, and numbers represent the mean and standard deviation of two biological replicates with at least 8 determinations total. (D,F) Comparison of RNA immunoprecipitated with TbRGG2-HTM (D) or MRB3010-PTP (F) grown with or without tet to induce MRB7260 RNAi. Fold change with MRB7260 knockdown represents the RNA levels detected in the RIP from tet-induced cells compared to the RIP from uninduced cells. RNA was detected using RIP mRNA primers described in A and primers designed to detect a subset of gRNAs using qRT-PCR. RNA levels were standardized against 18S rRNA and numbers represent the mean and standard deviation of two biological replicates with at least eight determinations total.