Elongator protein 3b negatively regulates ribosomal DNA transcription in african trypanosomes

Abstract

Eukaryotic cells limit ribosomal DNA (rDNA) transcription by RNA polymerase I (RNAP-I) to maintain genome integrity. African trypanosomes present an excellent model for studies on RNAP-I regulation because they possess a bifunctional RNAP-I and because RNAP-II transcription appears unregulated. Since Elp3, the catalytic component of Elongator, controls RNAP-II transcription in yeast and human cells, we predicted a role for a trypanosome Elp3-related protein, ELP3a or ELP3b, in RNAP-I regulation. elp3b null and conditional strains specifically exhibited resistance to a transcription elongation inhibitor, suggesting that ELP3b negatively impacts elongation. Nascent RNA analysis and expression of integrated reporter cassettes supported this interpretation and revealed negative control of rDNA transcription. ELP3b specifically localized to the nucleolus, and ELP3b loss rendered cells hypersensitive to DNA damage and to translation inhibition, suggesting that anti-Elongator function was important to maintain genome integrity rather than to modulate ribosome production. Finally, ELP3b displayed discrimination between RNAP-I compartments in the same cell. Our results establish ELP3b as a major negative regulator of rDNA transcription and extend the roles of the Elp3-related proteins to RNAP-I transcription units. ELP3b is also the first trypanosome protein shown to distinguish between rDNA and variant surface glycoprotein transcription within different RNAP-I compartments.

Fig. 1.

Phylogenetic and sequence analysis of Elp3 orthologues. (A) ELP3a and ELP3b were identified in T. brucei (Tb), T. cruzi (Tc), and Leishmania major (Lm). The unrooted neighbor-joining tree was generated using CLUSTAL 1.8X and TreeView. Where excellent (≥99.9%, open circles) or very good (≥90%, closed circles), branching confidence is indicated. Hs, Homo sapiens; Xl, Xenopus laevis; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tt, Tetrahymena thermophila; At, Arabidopsis thaliana. All accession numbers are indicated. The GeneIDs for the T. brucei proteins are as follows: TbELP3a, Tb927.8.5770; TcELP3a, Tc00.1047053503851.10; LmELP3a, LmjF16.0240; TbELP3b, Tb927.8.3310; TcELP3b, Tc00.1047053509769.110; LmELP3b, LmjF23.1350. (B) Schematic representation of the predicted structures of T. brucei ELP3a and ELP3b compared with human Elp3. The radical SAM domains (black boxes) and GNAT-type acetyltransferase domains (gray boxes) are indicated with the FeS cluster and motif A, respectively. The sequences were aligned using ClustalW, and the conserved domains (boxed) are indicated. Residues that are shared between all three proteins are white on a black background, and residues shared among any pair of proteins are on a gray background. Arrowheads indicate the Cys residues that form part of the FeS cluster. Asterisks indicate the conserved residues of motif A, QHXGXG, in all Elp3 orthologues.

Fig. 2.

ELP3b loss is associated with unstable resistance to transcription inhibition and hypersensitivity to a DNA-damaging agent. (A) Southern blot indicating ELP3a disruption. Genomic DNA was digested with HindIII. The map details the HindIII sites (h), deleted regions, and probes used (horizontal bars). The deleted region was replaced with the selectable marker BLE, HYG, or PAC; the HYG and PAC cassettes each contain a HindIII site. The elp3aPAC/HYG strain was used for the assay shown in panel F. (B) Southern blot indicating ELP3b disruption. The deleted region was replaced with the selectable marker PAC or BLA. Other details are as in panel A. (C) Population doubling time in wild-type strain and elp3a, elp3b, and elp3a/elp3b strains. elp3b* and elp3a/elp3b* strains were maintained in culture for more than 8 weeks. Standard deviations are indicated, and P values were derived using an unpaired Student t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. (D) Half-maximal effective concentrations (EC₅₀) for 6AU. All data were derived from two wild-type samples and two independent null strains. Other details are as in panel C above. (E) 6AU sensitivity (50 μg ml⁻¹). Other details are as in panel C above. (F) EC₅₀ for phleomycin. Other details are as in panel C above.

Fig. 3.

ELP3 downregulation phenocopies ELP3b knockout. (A) Western blotting with anti-GFP confirmed Tet-on (1 μg ml⁻¹) regulation of ^GFPELP3b in an elp3b background. The lower panel shows an equivalent Coomassie blue-stained gel as a loading control. Data from one representative cell line are shown. (B and C) Population doubling times (B) and 6AU (50 μg ml⁻¹) sensitivities (C) in wild-type (WT) and ^GFPELP3b strains. (D) Methyl methanesulfonate (MMS; 0.0004%) sensitivity in ^GFPELP3b strains (EC₅₀: +Tet, 0.00036% ± 0.000015%; −Tet, 0.00024% ± 0.000016%). The −Tet, ^GFPELP3b-depleted population was assessed 4 to 7 days after Tet removal. Data in panels B to D were derived from four independent clones. Standard deviations are indicated, and P values were derived using a paired Student t test. ***, P < 0.001; **, P < 0.01.

Fig. 4.

ELP3b negatively regulates transcription elongation at rDNA loci. (A) Schematic of a T. brucei rDNA transcription unit and the location of the R1 to R5 probes (horizontal bars) for nascent transcript analysis. Each rDNA unit is approximately 10 kbp in length (55). The promoter (flag) and the rDNA subunit coding regions are indicated. (B) Transcription run-on analysis during depletion of ^GFPELP3b. Phosphorimager signals were corrected against β-tubulin transcript abundance and expressed relative to the +Tet value (set to 1). The inset shows a sample slot blot. V2, VSG2; βT, β-tubulin. VSG2 is a single-copy gene ∼60 kbp from its promoter that is transcribed by RNAP-I. The spliced-leader RNA (SL-RNA) is derived from a tandem gene array and contributes a fragment that is trans-spliced to the 5′ end of every mRNA in trypanosomes. This RNA and β-tubulin, used as a loading control and also derived from a tandem gene array, are transcribed by RNAP-II. 5S rRNA is also derived from a tandem gene array and is transcribed by RNAP-III. Data were derived from four independent ^GFPELP3b strains. Error bars represent one standard deviation, and P values were derived using a paired Student t test. *, P < 0.05.

Fig. 5.

ELP3b suppresses transcription of an mRNA reporter integrated at an rDNA locus. (A) Schematic of a T. brucei rDNA transcription unit showing the location of the neomycin phosphotransferase (NPT) reporter. The flanking 5′-procyclin and 3′-aldolase untranscribed regions are represented by gray boxes. (B) Northern analysis of NPT mRNA expression following ^GFPELP3b depletion. One representative Northern blot is shown. An ethidium bromide-stained gel is included to show loading. Phosphorimager signals were processed as described for Fig. 4B. Data were derived from three independent ^GFPELP3b (rDNA::NPT) strains. Error bars represent 1 standard deviation, and P values were derived using a paired Student t test. *, P < 0.05.

Fig. 6.

Adaptation to the elp3b defect involves downregulation of rRNA transcription. (A) Schematic of a T. brucei rDNA transcription unit reproduced from Fig. 4A. (B) Transcription run-on analysis of adapted elp3b* null strains. Data were derived from two independent clones. Other details are as in Fig. 4B, except that corrected values are expressed relative to the wild type (WT). (C) Transcription run-on analysis of elp3a null strains. Data were derived from four independent clones. Other details are as in panel B above.

Fig. 7.

ELP3b localizes to the nucleolus but is not detected in the ESB in bloodstream-form T. brucei. (A) Western analysis of strains used for ELP3 localization studies. Blots were incubated with anti-MYC or anti-GFP. A Coomassie blue-stained gel is included to show loading. Predicted molecular masses of the fusions: ^GFPELP3a, 104 kDa; ^MYCELP3b, 77 kDa; ^GFPRPB6z, 42 kDa. The lower anti-GFP panel is a shorter exposure. (B) Dual localization of ^GFPELP3a and ^MYCELP3b. The effect of actinomycin D treatment is also shown. The regions outlined in the phase images indicate the regions shown in the immunofluorescence panels. The nucleus (n) and kinetoplast (k) were stained with the DNA intercalating dye 4′,6-diamidino-2-phenylindole (DAPI), and these images were merged with the immunofluorescence images. Bar, 5 μm. (C) ^MYCELP3b localizes specifically to the nucleolus but not to the smaller ESB (arrowhead), as revealed by ^GFPRPB6z. (D) Colocalization of ^GFPELP3b and the nucleolar protein, NOG1, through the cell cycle. G₁/S cells have a single nucleolus, while a second nucleolus can be seen during G₂/M (identified by two kinetoplasts). Postmitotic (Post-M) cells have a single nucleolus in each nucleus. Other details are as in panel B above.

Fig. 8.

ELP3a occupies nuclear territories distinct from those occupied by the transcription machinery. (A) Western analysis of strains used for ELP3 localization studies. Predicted molecular masses of the fusions: ^6MYCRPB6z, 24 kDa; ^6MYCRPB6, 25 kDa; ^GFPRPB6, 43 kDa; SNAP42^12MYC, 60 kDa; RPC160^12MYC, 188 kDa. (B) ^GFPELP3a and the tagged polymerase subunits occupy distinct nuclear territories. ^6MYCRPB6z, RNAP-I; ^6MYCRPB6 and SNAP42^12MYC, RNAP-II (colocalization of these factors is associated with sites of SL-RNA transcription [30, 52]); RPC160^12MYC, RNAP-III. (C) Localization of ^GFPELP3a and SNAP42^12MYC through the cell cycle. (D) ^GFPELP3a shows partial colocalization with NUP1. Other details are as in Fig. 7B.

Fig. 9.

ELP3b loss is associated with hypersensitivity to translation inhibition. (A) Total RNA from the wild type and pairs of elp3a, elp3b, and elp3a/elp3b null strains was fractionated by agarose-formaldehyde (upper panel) or polyacrylamide-urea (lower panel) gel electrophoresis. (B) Northern analysis of steady-state rRNA transcripts following depletion of ^GFPELP3b. One representative blot is shown. An ethidium bromide-stained gel is included to show loading. Phosphorimager signals were processed as described for Fig. 4B. Data were derived from four independent ^GFPELP3b strains. (C) EC₅₀ values for G418 and ^GFPELP3b strains, + and −Tet. −Tet cultures were maintained in Tet-free medium for 3 days prior to G418 treatment. Data were derived from four independent ^GFPELP3b strains. Standard deviations are indicated, and P values were derived using a paired Student t test. ***, P < 0.001.