DRBD3 regulates long non-coding RNA abundance and cryptic splice site selection in trypanosomes

Figures

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  Fig. 1. Effects of DRBD3 depletion on the abundance of lncRNAs. A Volcano plot of differential gene expression in DRBD3-depleted bloodstream and procyclic trypanosomes compared to control cells. Blue dots correspond to downregulated mRNAs, orange dots indicate upregulated mRNAs and green dots represent lncRNAs. DRBD3 transcript is shown as a black circle. Thresholds for differential abundance were |log2 fold change (FC)|≥ 1.0 and false discovery rate (FDR) ≤ 0.01. B Bar plot indicating relative proportions of up- and downregulated mRNAs and lncRNAs in DRBD3-, ZC3H41- and DRBD18-depleted cells. C Venn diagram of regulated lncRNAs detected upon depletion of DRBD3 (orange), ZC3H41 (purple) and DRBD18 (green). Numbers indicate unique and common lncRNAs between the datasets. D Boxplots representing length, abundance and association with polysomes of DRBD3-regulated lncRNAs (boxes, IQR; waists, medians; whiskers, ± 1.5 IQR; dots, outliers). TPM, transcripts per million; up, upregulated lncRNAs; down, downregulated lncRNAs. Two-tailed Mann–Whitney U-tests were used to assess whether there were significant differences between regulated and all annotated lncRNAs. *, p < 5 × 10−3; **, p < 5 ×…

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  Fig. 2. DRBD3-dependent regulation of representative lncRNAs. A grumpy; B TblncRNA-23; C KS17gene_6446a; D KS17gene_3091a; E KS17gene_1751a. Left panels, RNA-seq coverage plots of lncRNAs and genomic neighbors in control (blue) or DRBD3-depleted (orange profiles) bloodstream and procyclic trypanosomes. Average read counts across replicates were obtained using sliding windows (bin size, 100 bp; step, 10 bp) and normalized to library size (RPBinM, reads per bin per million mapped reads). Profiles corresponding to reads containing the spliced-leader sequence (SL) are also shown. Open-reading frames are represented as thick, filled boxes, while untranslated regions are depicted as thin, empty boxes (blue, genes in the Watson [+] strand; orange, genes in the Crick [–] strand). Long non-coding RNAs are shown as thin green boxes, and pseudogenes as thick grey filled boxes. Black arrows indicate the direction of transcription. Relevant lncRNA names are highlighted in bold, and their positions indicated by orange vertical arrows above the corresponding coverage profiles. RPBinM values for TblncRNA-23 procyclic coverage plots are displayed on a log10 scale. Right panels, validation of lncRNA and…

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  Fig. 3. Association of DRBD3 with regulated lncRNAs and effect of DRBD3 depletion on the expression of neighboring genes. A RNA immunoprecipitation followed by quantitative RT-PCR. Fold-enrichment of target transcripts was calculated after comparing to mock immunoprecipitations carried out using normal rabbit serum and normalizing to actin mRNA. Values are expressed as the mean ± s.e.m. (n = 3). Transcripts located upstream of lncRNAs are highlighted in bold; transcript systematic identifiers are shown without the ‘Tb927.’ prefix. Two-tailed, one-sample t-tests assessed whether mean values differed significantly from log2 = 0 (no enrichment, dashed line). Only statistically significant changes are marked (*, p < 5 × 10−2; **, p < 5 × 10−3). B-H western blot analysis of 4xTy-tagged proteins encoded by neighboring genes in bloodstream (B and H) and/or procyclic extracts (B-H). α-tubulin (panels B -procyclic-, D and E), RRP4 ([53], panels B -bloodstream-, C, F and G) or p22 ([54], panel H) were used as loading controls. I quantification of 4xTy- tagged proteins levels in three independent experiments; *, p < 5 × 10−2; **, p < 5 × 10−3 (two-tailed, one-sample t-tests vs. log2 = 0, dashed…

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  Fig. 4. DRBD3 regulates the alternative trans-splicing of the transcript encoding the tRNA-import complex protein Tb927.11.12740. A Reads containing the spliced-leader at the internal SAS (splicing-acceptor site within the ORF) or the 5’ SAS (defining the longest transcript isoform). Values (reads per million mapped reads, RPM) are expressed as the mean ± s.e.m. (n = 3); all edgeR FDR values < 5 × 10−4. B Western blot analysis of Tb927.11.12740-TAP protein levels in uninduced vs DRBD3-silenced trypanosomes; α-tubulin was used as a loading control. The arrow indicates a truncated Tb927.11.12740-TAP species; the full-length protein has an estimated molecular mass of ~ 60 kDa. C Quantification of Tb927.11.12740-TAP depletion (full-length species). Log2FC (western signal in RNAi vs uninduced) values are shown as mean ± s.e.m. (n = 3). *, p < 5 × 10−3 (two-tailed, one-sample t-test vs log2 = 0, dashed line).

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  Fig. 5. DRBD3 regulates the expression of intron-containing genes. A Quantification of split-reads corresponding to PAP1, DBP2B and RBP20 transcripts in control vs DRBD3-depleted trypanosomes. Reads per million mapped reads (RPM) values are shown as mean ± s.e.m. of RNA-seq replicates (n = 3); *, p < 5 × 10−2, **, p < 5 × 10−3 (two-tailed, independent t-tests). B RT-PCR splicing assay of the RBP20 transcript. Diagrams corresponding to unspliced (top) or spliced (bottom) transcripts are shown on the right; arrows represent the oligonucleotides used for PCR amplification. Actin (ACT) transcript was amplified using specific oligonucleotides to ensure equivalent amounts of input RNA in both samples. C and D coverage plots corresponding to the PAP1 (procyclic) or RBP20 (bloodstream) loci; the arrow in D points to spliced-leader reads at the 5’-end of RBP20 exon 2. See Fig. 2 legend for details and Fig. S7 for additional plots. E Quantification of reads containing the spliced-leader at the 5’-end of RBP20 exon2. RPM values are shown as mean ± s.e.m. of RNA-seq replicates (n = 3); both edgeR FDR values were < 5 × 10−15. F Expression of TAP-PAP1, 4xTy-DBP2B and 4xTy-RBP20 tagged proteins in…