Combinatorial interplay of RNA-binding proteins tunes levels of mitochondrial mRNA in trypanosomes

Figures

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  FIGURE 1.. MRP1 iCLIP analysis. (A) Schematic depiction of the MRP1 iCLIP workflow to purify UV-crosslinked RNA-MRP1 complexes and sequence the mRNAs. (B) Schematic representation of the problem of iCLIP mapping assignment ambiguity due to the presence of editing intermediates in the transcriptome. Many of the approximately 30 to 50 nt iCLIP tags can be attributed to a partially edited or to a pre/fully edited version of a transcript. Therefore, the editing state of a transcript from which an iCLIP tag was derived cannot be unambiguously defined. Small blue boxes depict editing events. (C) Illustration of the MRP1 iCLIP read mapping strategy. Preprocessed iCLIP reads were mapped against maxicircle-encoded mRNAs. (Top) iCLIP reads mapped to reference sequences existing on the maxicircle; namely, never-edited and pre-edited mRNA sequences. (Bottom) iCLIP reads mapped to fully edited sequences of edited mRNAs. Pan-, minimally, and never-edited mRNAs are marked in blue, gray, and black, respectively.

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  FIGURE 2.. MRP1 iCLIP results. (A) Percentage of total MRP1 iCLIP tags obtained from each of the three mRNA categories. (B) Transcript length-normalized percentage of total MRB1 iCLIP tags obtained from each of the three mRNA categories. (C) Fold enrichment of MRB8170, MRB4160, and MRP1 iCLIP tags relative to mapped mRNA-seq read counts for three mRNA categories. (D) Genomic browser snapshots of MRP1 and MRB8170 presence on the pre-edited forms of ND8 (pan-edited) and CYB (minimally edited), and never-edited ND4 mRNAs. White lines show the approximate distribution of editing sites on ND8 and CYB. See also Supplemental Figures S1–S5.

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  FIGURE 3.. Comparison of MRP1 and MRB8170 association with mRNAs. (A) Plots comparing the total number of iCLIP tags of each of nine pan-edited mRNAs from MRB8170 IPs to those of MRB4160 IPs, and MRB8170 iCLIP tags to total mRNA-seq data. Pearson correlation coefficients are (r) indicated. (B) As in B, from MRP1 iCLIP, MRB8170 iCLIP, and total mRNA-seq data. (C) Correlation between total MRP1 iCLIP tags (y-axis) of each pan-edited transcript and its respective number of U insertions and deletions required to generate the fully edited, translatable sequence (x-axis). Pearson correlation coefficients (r) are indicated. (D) Plots comparing the total number of iCLIP tags of the combined minimally and never-edited mRNAs from MRB8170 and MRP1 iCLIP data. The Pearson correlation coefficient (r) is indicated. See also Supplemental Figure S6.

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  FIGURE 4.. RNA crosslinked peak profile comparison between MRP1- and MRB8170- associated mRNAs. (A) Genomic browser snapshots of MRP1 and MRB8170, as well as RNA-seq on the pre-edited forms of ND7 and COX3 (pan-edited), CYb and COX2 (minimally edited), and ND4 and ND5 (never-edited) mRNAs. The shaded peaks show the clustering of RNA crosslinked peaks at similar positions over mRNAs in MRB8170 and MRP1. (B) Genomic browser snapshots of MRB8170 and MRB4160, as well as RNA-seq on the pre-edited forms of ND7 and ND8 (pan-edited). The shaded peaks show the clustering of RNA crosslinked peaks at similar positions over mRNAs in MRB8170 and MRB4160.

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  FIGURE 5.. RIP-qPCR for MRP1 and TbRGG2. (A) MRP1 RIP-qPCR. Bar plots show the relative amount of representative pre- and never-edited mRNAs coimmunoprecipitated with MRP1. Abundance of each mRNA is presented relative to the abundance of the same mRNA recovered from 1% of input lysate. Two biological replicates (collected on different days) were performed in triplicate. Error bars show standard deviation. Table shows the averages and standard deviation of the tested mRNAs. (B) TbRGG2 RIP-qPCR. As in A.

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  FIGURE 6.. MRP1 and TbRGG2 recruited to 8170 on mRNAs in a class-specific manner. (A) Enrichment ratios of bound MRB8170 relative to bound MRP1 (y-axis) of each pan-edited, minimally edited, and never-edited mRNA. The first column includes all pan-edited mRNAs; these are affected by TbRGG2 depletion. The second column includes minimally and never-edited mRNAs; these are unaffected by TbRGG2 depletion (Mann–Whitney test; [**] P < 0.005; SD, standard deviation; CV, coefficient of variation). (B) As in A, except that the enrichment ratio compares bound MRB8170 relative to bound MRB4160. MRB4160 does not bind to never-edited mRNAs. (C) Relative amounts of MRP1 coimmunoprecipitated mRNAs in three different conditions using RIP-qPCR (Tukey's multiple comparison test, [*] P < 0.05, [***] P < 0.001, [****] P < 0.0001, biological replicates n = 2–3). Each mRNA abundance is presented relative to the abundance of the same mRNA recovered from 1% of input lysate. (D) Combinatorial interplay involving three RNA-binding proteins. In pan-edited transcripts, MRB8170 marks the pre-edited mRNAs and is followed by TbRGG2 to allow efficient progression of editing. Since TbRGG2 is nonessential for minimally and…