The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I

Figure 1.. Blocking RNA export has little effect on global patterns of nascent transcription, but increases the expression of procyclin-associated genes and downstream transcripts. (A) Scheme of the experimental approach: RNAi was induced by addition of tetracycline (+Tet) and 48 h later the block in export was monitored by polyA RNA FISH. Following a 10 min pulse with 5-EU, nascent RNA was extracted for sequencing. In the case of TbMex67, an RNAi cell line that also overexpressed a dominant negative (DN) isoform upon Tet addition was employed as well (TbMex67 RNAi/DN-OE). Representative pictures for FISH experiments from uninduced and induced TbMex67 RNAi/DN-OE are shown (scale bar, 5 μm). (B) Graphs represent growth curves of induced (+Tet) and non-induced (-Tet) RNAi cell lines (left panels) and RPM (reads per million) counts from nascent RNAs with Pearson correlation coefficients (right panels). (C) Differential expression analysis (DE-Seq) from nascent transcripts for induced and non-induced TbMex67 RNAi/DN-OE (n = 3). Gene IDs for PAGs (procyclin-associated genes) and GRESAGs (gene related to expression site-associated gene) from chromosomes 10 and 6 are indicated in green. Genes…

Figure 2.. Overexpression of TbMex67 DN results in polymerase I readthrough. (A) Patterns of nascent transcription and Pol I and H3 ChIP-seq mapped reads visualized in Genome Browser for a region on chromosome 10 where procyclins and PAGs are encoded. For the nascent transcripts, reads per kb per million (RPKM) were split for the forward (FW) and reverse (REV) strands. Reads for induced (+Tet) and non-induced (–Tet) TbMex67 RNAi/DN-OE are shown. For ChIP-seq of Pol I and H3, the ratios of induced to non-induced (+/– Tet) reads are shown on a log2 scale. The track for H3K4me3 (trimethylation of lysine 4 of histone 3) ChIP-seq marks transcription start sites (29). The green tracks and arrow show the directionality of the Pol I polycistronic transcription unit (PTU) while the blue tracks and arrow show the directionality of the Pol II PTU. (B and C) Western blot for whole cell lysates (left) and subcellular fractionation (right), WCL: whole cell lysate, C: cytoplasm, N: nucleus) from induced and uninduced cells. The antibodies that were used are indicated below each panel. (D) Fluorescence microscopy for detection of L1C6 and Pol I from induced and uninduced cells. (E) Quantification of the…

Figure 3.. TbMex67 N-terminal domain is essential and required for nuclear localisation. (A) Scheme of TbMex67 modular architecture and the different deletion and point mutants (CCCH‐type zinc finger domain (CCCH), leucine‐rich repeats (LRR), nuclear localisation signal (NLS)). (B–D) Western blot (B), growth curves (C) and immunofluorescence of TbMex67 and polyA RNA FISH (D) upon RNAi and complementation with the different variants (scale bar, 5 μm). (E) TbMex67 quantification of nuclear/cytoplasmic ratio in the presence of actinomycin D (2 h at 5 μg/ml). Average values are shown with standard deviations and P-values, determined using a Mann–Whitney U test. Significant P-values are indicated by the asterisks above the graphs (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05). Representative pictures for TbMex67 immunofluorescence from control and actinomycin D treated cells are shown.

Figure 4.. TbMex67 association with chromatin depends on its N-terminal domain. (A and B) TbMex67 distribution along genes obtained by TbMex67 ChIP-seq. C) Metaplots and mapped reads for TbMex67 ChIP-seq at regions that are specific for each RNA polymerase. Pol I and II ChIP-seq reads (normalised to input), as well as nascent transcripts (RPKM), are also shown. A bed file containing Pol III-transcribed regions was utilised to compensate for the lack of Pol III ChIP. Metaplots show the coverage of TbMex67 ChIP normalised to input from uninduced and induced cells at regions transcribed by each polymerase.

Figure 5.. TbMex67 shares genomic occupancy and protein complexes with TbRRM1 and histone H3. (A) Genome-wide comparison of TbMex67, histone H3 and TbRRM1 from induced and uninduced cells. (B) Immunofluorescence of TbMex67 and HA-TbRRM1 (scale bar, 5 μm). (C) Coimmunoprecipitation of TbMex67 and H3 with HA-TbRRM1. (D) Immunoprecipitation of TbMex67-PTP.

Figure 6.. Overexpression of TbMex67 DN induces accumulation of R-loops and γH2A nuclear foci and inhibits DNA replication. (A) Visualisation of the nucleolar marker L1C6 and R-loops with quantification of fluorescence intensity across the white arrow. R-loops were detected by staining with the antibody S9.6. (B) Quantification of R-loops (S9.6-positive foci), following transcription inhibition by alpha-amanitin (20 h at 100 μg ml−1), CX5461 (3 h at 3 μM) and actinomycin D (2 h at 5 μg ml−1) or RNase H treatment (20 min at 37ºC). (C) Visualisation of R-loops and γH2A in induced (+Tet) and uninduced (-Tet) TbMex67 RNAi/DN-OE cells (Scale bar, 1 μm). (D–F) Quantification of S9.6-positive foci (D), γH2A (E) and EdU incorporation (F) in induced and uninduced cells. Cell lines over-expressing TbRNaseH1 (OE) or a TbRNaseH1 single knockout (+/-) were used as controls. Representative images are shown. G) Quantification of γH2A-positive loci upon induction of TbMex67 RNAi/DN-OE cells and/or addition of 3 μM CX5461. Average values are shown with standard deviation and p-values, determined using a Mann–Whitney U test. Significant p-values are indicated by the asterisks above the graphs (****P< 0.0001;…

Figure 7.. Working model. Mapped reads for nascent transcripts; Pol I, H3 and TbMex67 ChIP-seq and R-loops at the procyclin locus on chromosome 10 from induced (+Tet) and uninduced (–Tet) TbMex67 RNAi/DN-OE cells. R-loops were identified by S9.6 DRIP-seq. Model depicting the role of TbMex67 in preventing readthrough by Pol I. In the absence of the CCCH domain, the protein shows reduced association with chromatin and the nuclear pore and likely sequesters binding partners. NUPS: nucleoporins, Pol I: RNA polymerase I, Pol II: RNA polymerase II. Histone H3 depletion aids Pol I progression. Since the export function of TbMex67 is also impaired, the antisense transcripts retained in the nucleus give rise to R-loops that trigger DNA damage.