An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

Figures

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  Fig. 1. Extraordinarily high levels of splicing occur at the active VSG ES, which is necessary for its transcription. a Estimated mRNA transcripts produced per hour from an active VSG gene, an average of all Pol II-transcribed genes (Pol II), or a tubulin gene (Tub) in bloodstream form (BF) T. brucei. This is compared with mRNA from a procyclin (EP or GPEET) gene in procyclic form (PF) T. brucei. b Estimated difference in splicing rate (fold difference) at the active VSG gene or an average ES gene (BES) compared with an average of all Pol II-transcribed genes. The data used to calculate the values presented in panels (a, b) are shown in Supplementary Tables 1 and 2. c Representative fluorescence microscopy images of RNA-FISH detecting the U2 snRNA (green) or VSG pseudogene (Ψ) transcript (magenta) specific for BES1 in BF T. brucei cells. The nucleus is indicated with a dashed line, scale bar = 1 μm. A line profile displaying the U2 snRNA and VSGΨ signal intensities is shown, with fluorescence indicated in arbitrary units (arb. units). d Quantitation of the percentage (%) of cells with MS2 RNA-FISH signal in cells with different genomic loci marked with MS2 repeats, at different time…

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  Fig. 2. Identification of a Spliced Leader Array Body (SLAB) which frequently associates with the active ES. a Fluorescence microscopy imaging of a triple epitope-tagged BF T. brucei cell line expressing the SLAB components SLAP1, SNAP3 and VEX1. Signals in the nucleus (dashed line) correspond to mNG::SLAP1 (SLAP-mNG, green), SNAP3::6xHA (SNAP3-HA, magenta) and VEX1::12xmyc (VEX1-myc, red). Scale bar = 1 μm. b Schematic of SLAP1 protein motifs identified using PFAM and disordered domains with PONDR. c Quantitation of the percentage (%) of BF T. brucei cells with different numbers of SLAP1 foci (1–3), visualised using an mNG::SLAP1 cell line. Cells at different cell cycle stages with various numbers of kinetoplasts (K), elongated kinetoplasts (eK) or nuclei (N) were analysed. Values are the averages of three biological replicates with error bars indicating ± SD. For 1K1N n = 218, 1eK1N n = 57, 2K1N n = 38 2K2N n = 34. Values obtained from each replicate are shown with circles. d SR-SIM imaging of individually epitope-tagged BF T. brucei cell lines expressing the SLAB1 components SNAP3, VEX1 and SLAP1 in the nucleus (dashed line). Cell lines express: SNAP3::6xHA (SNAP3-HA, green),…

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  Fig. 3. SLAP1 regulates SL-RNA production with knockdown resulting in an ES transcription block. a SR-SIM imaging of a BF T. brucei cell line with nuclear signals corresponding to mNG::SLAP1 (SLAP1 mNG, green) or SNAP3::6xHA (SNAP3-HA, red), with the SL-RNA intron (magenta) visualised with RNA-FISH. SLAP1 RNAi was induced for the time in hours (h). b) RNA quantitation using qPCR from cells in (a) after induction of SLAP1 RNAi for the time in hours (h). Nascent RNA is from the promoter (Prom) or telomere (Telo) of BES1, or from the actin or rRNA loci. Average values are from three biological replicates with error bars indicating ± SD. **P ≤ 0.01; *P ≤ 0.05. For SLAP1 mRNA, P = 0.006, BES1 telomere pre-mRNA P = 0.02 and actin pre-mRNA P = 0.0051. c Fluorescence microscopy imaging of a BF T. brucei cell line, with mNG::SLAP1 (SLAP1 mNG, green) and SNAP3::6xHA (SNAP3-HA, red) signal shown, with VSGΨ transcript (magenta) detected with RNA-FISH. SLAP1 RNAi was induced for the time in hours (h). d Quantitation of the percentage (%) of cells in (c) with VSGΨ RNA-FISH signal after induction of SLAP1 RNAi for the time in hours (h). Values are the average of three biological replicates, with error…

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  Fig. 4. Identification of a novel NUFIP body. a Schematic of NUFIP body components: NUFIP, RBP34, ZNHIT3 and the newly identified NufB1 and NufB2 proteins. Relevant protein domains and motifs were identified using PFAM, and disordered domains with PONDR (coloured boxes). b SR-SM imaging of a BF T. brucei cell line with three NUFIP components epitope-tagged. Signals in the nuclei (dashed lines) correspond to NUFIP::mNG (NUFIP-mNG, green), mCh::NufB1 (NufB1-mCH, red) and NufB2::6HA (NufB2-HA, magenta), with DNA stained with DAPI (blue). The insets show further magnification of the foci, illustrating the ‘shell’-like structure. Scale bar = 1 μm for main images, and 200 nm for the insets. c As in (b), only showing SR-SM microscopy imaging of a BF cell line with two NUFIP body components epitope-tagged: NufB2::6HA (NufB2-HA, magenta) and RBP34::mNG (RBP34-mNG, green). d As in (b), but showing SR-SM microscopy imaging of a BF cell line with two NUFIP body components epitope-tagged: NufB2::6HA (NufB2-HA, magenta) and ZNHIT3::mNG (ZNHIT3-mNG, green).

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  Fig. 5. The NUFIP body associates with the active ES in BF T. brucei. a Quantitation of the percentage (%) of BF T. brucei cells with different numbers of NUFIP foci (1–4) monitored using SR-SIM imaging. Different cell cycle stages are shown, with the number of kinetoplasts (K), elongated Kinetoplast (eK) or nuclei (N) indicated. Values are the averages from three biological replicates with error bars indicating ± SD. The values from individual replicates are shown with circles. For 1K1N n = 333, 1eK1N n = 76, 2K1N n = 49 and 2K2N n = 38. b Widefield microscopy imaging of a BF T. brucei cell line with the SLAB and NUFIP bodies visualised, combined with RNA-FISH detecting BES1 nascent RNA using a VSG pseudogene probe (VSGΨ, white). This line expresses mCh::SLAP1 (SLAP1-mCh, red) and NUFIP::mNG (NUFIP-mNG, green). The nucleus (dashed line) is stained with DAPI (blue). Scale bar = 1 μm. c SR-SIM imaging of a nucleus (dashed line) from BES1 expressing BF T. brucei which has the NUFIP body components NUFIP (NUFIP-mNG, green) or NufB2 (BUFB2-mNG, green) epitope-tagged with mNeon green. BES1 nascent transcript is detected with a VSG pseudogene (VSGΨ, magenta) probe. Alternatively, a BES2…

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  Fig. 6. A subset of BF T. brucei cells have a Cajal body that associates with the active ES. a SR-SIM imaging of a BF T. brucei cell line expressing two conserved Cajal body markers which are epitope-tagged: mNG::NOP56 (NOP56-mNG, green) and 3xHA::fibrillarin2 (fibrillarin2-HA, red). The Cajal body is indicated with a light-blue arrowhead. b Quantitation of the percentage (%) of BF T. brucei cells with 1–3 Cajal bodies, as determined by fluorescence microscopy imaging of a cell line expressing mNG::NOP56. Cells throughout the cell cycle containing different numbers of kinetoplasts (K), elongated kinetoplasts (eK) or nuclei (N) were monitored. Average values are shown from three biological replicates, with error bars, indicating ±SD. The values from individual replicates are shown with circles. For 1K1N n = 284, 1eK1N n = 120, 2K1N n = 40 and 2K2N n = 30. c Fluorescence microscopy imaging of a cell line expressing the Cajal body component mNG::NOP56 (NOP56-mNG, green) and the Pol I subunit TdT::RPA2 (RPA2-TdT, red). The nucleolus is visualised with an anti-L1C6 antibody (magenta), detecting an uncharacterised nucleolar protein. The Cajal body is indicated with a light-blue arrowhead and…

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  Fig. 7. An assembly of nuclear bodies associates with the active ES in BF T. brucei. a Simultaneous visualisation of the ESB, SLAB and NUFIP bodies using SR-SIM imaging of a triple epitope-tagged BF T. brucei cell line. This cell line expresses mNG::RPA2 (RPA2-mNG, green), mCh::SLAP1 (SLAP-mCh, red) and NufB2::6HA (NufB2-HA, magenta). The ESB is indicated with a white arrowhead within a nucleus (dashed line) stained with DAPI (blue). Scale bar = 1 μm. b Quantitation of the distance between the ESB (visualised with RPA2) and the SLAB (visualised with SLAP1) compared with the distance between the ESB and the NUFIP body (visualised with NufB2). Data was of the cell line shown in (a) using SR-SIM imaging. The scatter plot is divided into four quadrants which are coloured according to whether the different bodies in the cells are within 500 nm of each other. Quantitation of the percentage (%) of cells within each coloured quadrant is on the right. Example images of cells from each quadrant are shown in Supplemental Fig. 10. N = 110 cells in G1 (1K1N) from two biological replicates. c Simultaneous visualisation of the Cajal, SLAB and NUFIP bodies using SR-SIM imaging of a triple epitope-tagged…

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  Fig. 8. The highly SUMOylated focus is in close proximity to the ESB. a SR-SIM imaging of the ESB (Halo::RPA2, magenta) with the highly SUMOylated focus (HSF; α-SUMO antibody, green) (upper panel) or the ESB (mNG::RPA2, green) with VEX2::6HA (magenta) (lower panel) in BF T. brucei. The ESB is indicated with a white arrowhead. b Combined RNA-FISH detecting nascent transcripts from the active BES1 promoter (BES1-Prom-24xMS2, red) and the active BES1 telomere (VSGΨ, magenta) with visualisation of the ESB (mNG::RPA2, green; upper panel) or the HSF (α-SUMO antibody, green; lower panel) using SR-SIM imaging. The ESB is indicated with a white arrowhead. c Violin plot showing quantitation of the distances of the ESB (RPA2) to either VEX2 or the HSF using microscopy images shown in (a) or the distances of nascent RNA from either the active BES1 promoter or telomere to either the HSF or ESB (RPA2) using microscopy images shown in (b). The number of 1K1N cells counted for each quantitation was VEX2-RPA2 n = 52, HSF-RPA2 n = 184, BES1 RNA-HSF n = 120, BES1 RNA-RPA2 n = 189. Cells with BES1 transcripts were only quantitated when both transcripts (promoter and telomere) were present. Measurements were…

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  Fig. 9. The SLAB, NUFIP and Cajal bodies are present in PF T. brucei, with the SLAB and NUFIP bodies frequently associated with each other, but not with the EP1 procyclin locus. a The SLAB was investigated using microscopy imaging of a PF T. brucei line expressing the epitope-tagged SLAB components mNG::SLAP1 (SLAP-mNG, green), SNAP3::6HA (SNAP3-HA, magenta) and VEX1::12myc (VEX1-myc, red). The NUFIP body was investigated using SR-SM imaging of a PF cell line expressing epitope-tagged NUFIP body components NUFIP::mNG (NUFIP-mNG, green) and NufB2::6HA (NufB2-HA, red). The inset image shows a magnification of the NUFIP body. The Cajal body was investigated with a PF T. brucei line expressing Cajal body components mNG::NOP56 (NOP56-mNG, green) and 3xHA::fibrillarin2 (fibrillarin2-HA, red). The arrowhead indicates the Cajal body. b SR-SIM imaging of RNA-FISH visualising nascent MS2 transcripts expressed from 24 MS2 repeats inserted in the EP1 procyclin locus (EP1-MS2, red) combined with imaging of NUFIP::mNG (NUFIP-mNG) or mNG::SLAP1 (SLAP-mNG, green). A schematic of the EP1 procyclin locus containing MS2 repeats (hatched box) is shown above, with the promoter indicated with a flag and…

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  Fig. 10. Model of the architecture of the Expression Site nuclear body assembly in BF T. brucei. Schematic representation of a BF T. brucei nucleus (light blue) with the nucleolus indicated (dark blue). Active and silent ESs are expanded in the green boxes. The active ES is associated with four discrete nuclear bodies: The ESB (green circle), the Cajal body (dark blue circle), the SLAB (red circle) and the NUFIP body (purple circle). The ESB contains Pol I and VEX2, and shows partial overlap with a highly SUMOylated focus (SUMO). The SLAB closely associates with the spliced leader (SL) RNA gene locus. The ESs contain various genes (white boxes) in addition to the telomeric VSG gene, with the promoters indicated with flags, transcription with a red arrow and telomere repeats with black triangles. These four nuclear bodies appear to often cluster in a single nuclear compartment which forms an ES nuclear body assembly (large light-blue circle with red dashed outline) satisfying the high demand for splicing at the active ES. Silent ESs are not associated with this ES nuclear body assembly and the low amount of nascent RNA generated from these ESs is not spliced efficiently.