Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2013 Dec;41(22):10135-49.
doi: 10.1093/nar/gkt770. Epub 2013 Sep 9.

Structure-function Analysis of Hmo1 Unveils an Ancestral Organization of HMG-Box Factors Involved in Ribosomal DNA Transcription From Yeast to Human

Affiliations
Free PMC article

Structure-function Analysis of Hmo1 Unveils an Ancestral Organization of HMG-Box Factors Involved in Ribosomal DNA Transcription From Yeast to Human

Benjamin Albert et al. Nucleic Acids Res. .
Free PMC article

Abstract

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of ∼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.

Figures

Figure 1.
Figure 1.
When expressed in S. cerevisae, UBF1 and UBF2 are nucleolar and increase nucleolar volume. (A) UBF1 and UBF2 fused to YFP and produced in yeast concentrate in a nuclear crescent shape structure. (B) The construct encoding the YFP-UBF1 fusion protein (green) was used to transform a strain producing a Hmo1-CFP fusion protein (blue) and an mRFP-Nop1 fusion protein (red), which define different subdomains of the nucleolus. UBF1, Hmo1and Nop1 fully co-localize. (C) A strain producing mRFP-Nop1 fusion protein (red) and GFP-Nup49, which reveals the nucleolus and nuclear, respectively, periphery was transformed with empty plasmid (Control), a plasmid over-expressing Hmo1 or UBF1 (not shown) or UBF2. (D) Nuclear and nucleolar volumes were imaged by confocal microscopy and quantified using automated detection software (32). Cumulative frequency plots of the nucleolar to nuclear volumes ratio show a decrease on Hmo1 overexpression [Two-sample Kolmogorov–Smirnov test (KS), P = 2.7e−06] and a significant increase on UBF1 (KS test, P = 1.6e−08) or UBF2 (KS test, P = 4.3e−07) expression relative to control. Scale bar is 5 µm.
Figure 2.
Figure 2.
UBF1 can stimulate growth of the rpa49Δ-hmo1Δ mutant. (A) UBF1 and UBF2 are expressed at similar level in budding yeast as estimated by western blot. Total proteins were extracted from WT strain bearing an empty plasmid (−), expressing UBF1 or UBF2. PGK1 was used as loading control. (B) UBF1, but not UBF2, partially complements the growth defect of rpa34Δ-hmo1Δ strain. WT, hmo1Δ and hmo1Δ-rpa34Δ double mutant expressing UBF1 or UBF2 were spotted on minimum media, and growth was scored after 3 days at 25°C. (C) UBF1 complements the essential functions of Hmo1 in rpa49Δ-hmo1Δ. Ten-fold serial dilutions of the double rpa49-hmo1 deleted strain expressing Hmo1, UBF1, UBF2 or bearing an empty vector (−) were spotted on plates containing 5-FOA to test for complementation (see ‘Materials and Methods’ section). (D) Double hmo1Δ-rps23bΔ deleted strain expressing Hmo1, UBF1 or an empty vector (−) were spotted on plates containing 5-FOA to test for complementation. Plates without FOA (−) were used as controls to confirm that similar numbers of cells were spotted.
Figure 3.
Figure 3.
UBF1 and Hmo1 stimulate rDNA transcription in budding yeast. (A) Ten-fold serial dilutions of the rpa49 deleted strain expressing Hmo1, UBF1, RPA49 or an empty vector (−) were spotted on plates containing 5-FOA and incubated at 25°C for 5 days to test for complementation of rpa49Δ growth defect. (B) In vivo labeling of newly synthesized RNAs. Two different clones of WT (lanes 1–2 and 7–8), rpa49Δ (lanes 3–4 and 9–10) and rpa49Δ strains expressing UBF1 (lanes 5–6 and 11–12) were grown to an OD600 of 0.8. Cells were then pulse-labeled with [8-3H] adenine for 12 min. Samples were collected and total RNAs were extracted, separated by gel electrophoresis. High molecular weight RNAs were resolved on an agarose gel (left panel, lanes 1–6), low molecular weight RNAs were resolved on a polyacrylamide gel (right panel, lanes 7–12). (C) Effect of Hmo1 and UBF1 overexpression on rDNA occupancy by Pol I. ChIP assays were performed on a strain with 25 rDNA copies expressing a HA-tagged version of the largest subunit of Pol I, Rpa190 and deleted for RPA49 (rpa49Δ, gray line). This strain is transformed with the appropriate plasmids allowing expression of Rpa49 (complemented WT, black line), overexpression of Hmo1 (rpa49Δ + Hmo1; red line) or overexpression of UBF1 (rpa49Δ + UBF1; green line). Pol I was pull-down by anti-HA antibodies. ChIP from untagged strains is used as background binding (no tag). DNA occupancy was defined by the ratio between the immunoprecipitation (IP) and the input signals. The bottom diagram shows the positions of the primers used for qPCR amplification on rDNA. The arrow denotes the start site and transcription orientation of the 35S rRNA gene.
Figure 4.
Figure 4.
UBF1 N-terminal domain is sufficient to rescue viability of rpa49Δ-hmo1Δ strain. (A) Schematic representation of UBF1. UBF1 depicted as an N-terminal dimerization domain (UBF-D) and five HMG Boxes (1–5). Truncated UBF1 constructions, Box1 and Box1-2, are shown by square brackets. (B) YFP-truncated proteins were co-expressed with CFP-Nup49 and mRFP-Nop1 fusion proteins that, respectively, delineate the nuclear periphery (blue) and the nucleolus (red). Truncated UBF1 constructions Box1 and Box1-2 localized in the yeast nucleolus. (C) UBF1-Box1-2 is sufficient to stimulate Pol I. Ten-fold serial dilutions of the rpa49Δ-hmo1Δ strain expressing the UBF1 truncation Box1, Box1-2, or Hmo1 or an empty vector (−) were spotted onto plates containing 5-FOA to test for complementation (see ‘Materials and Methods’ section).
Figure 5.
Figure 5.
Identification of a Hmo1 counterpart in S. pombe. (A) Schematic representation of Hmo1. The position of the consensus HMGB region B (black rectangle) is shown in the schematic representation of Hmo1 and the amino-acid compositions of UBF1 and Hmo1 are presented in the close-up view. Residues that are identical are written in red and conserved hydrophobic residues are written in blue. (B) Localization of the HMGB of Hmo1 (black rectangle) and in four S. pombe’s proteins (Spbc28f2.11, Spac10f6.08c, Spac4g9.11c, Spac57a10.09c). Number of amino acids of each protein is indicated. (C) Spbc28f2.11 localizes to the nucleolus when expressed in budding yeast. The four HMGB proteins from S. pombe were co-expressed in S. cerevisiae as YFP fusion proteins (green) with CFP-Nup49 (gray) and mRFP-Nop1 (red) fusion proteins to visualize the nuclear periphery and the nucleolus, respectively (D) Spbc28f2.11 can substitute for the essential function of Hmo1 in a rpa49Δ-hmo1Δ background. Ten-fold serial dilutions of cultures of the rpa49Δ-hmo1Δ mutant expressing Hmo1, one HMGB from S. pombe or an empty vector (−) were spotted on plates containing 5-FOA to test for complementation. (E) Spbc28f2.11 cannot substitute for the essential function of Hmo1 in a hmo1Δ-rps23bΔ background. Ten-fold serial dilution of cultures of the rps23bΔ-hmo1Δ mutant expressing Hmo1, Spbc28f2.11 or an empty vector (−) were spotted on plates containing 5-FOA to test for complementation.
Figure 6.
Figure 6.
Characterization of the Hmo1 counterpart in S. pombe. (A) Localization of Sp-Hmo1 in S. pombe. Sp-Hmo1 was produced as a C-terminal HA-tagged protein in S. pombe and detected by immunofluorescence (green). The nucleolus was revealed by immunodetection of the Gar2 protein (red) and DAPI labeled the nucleus (blue). Scale bar 5 µm (B) Sp-Hmo1 is required for 35S pre-rRNA accumulation. Total RNA was extracted from a WT strain and Sp-hmo1Δ (Δ) mutant cells and analyzed by northern blotting. Hybridizations revealed the precursors indicated on the left of each panel. (C) The Sp-hmo1Δ-Sp-rpa49Δ double mutant is not viable. After mating of haploid Sp-hmo1Δ and Sp-rpa49Δ strains and meiosis, growth of segregants bearing Sp-hmo1Δ (K) and Sp-rpa49Δ (U) was followed by tetrad analysis. WT and single mutants Sp-hmo1Δ and Sp-Rpa49Δ were selected with KAN (K) and URA3 (U); their frequencies are close to those expected, but no double mutant was isolated. (D) Overexpression of Sp-Hmo1 suppresses the growth defect of Sp-rpa49Δ at 25°C. The Sp-rpa49Δ mutant transformed with empty vector (−), Sp-RPA49 complementing the deletion (WT) or Sp-HMO1 driven from a strong promoter were grown for 4 days at 25°C or 30°C. (E) Accumulation of rRNA precursors was restored in the Sp-rpa49Δ mutant by overexpression of Sp-Hmo1. Total RNA was extracted and analyzed by northern blotting. Hybridizations revealed the precursors indicated on the left of each panel.
Figure 7.
Figure 7.
Hmo1 is unable to fulfill UBF1 essential function(s) in human cells. (A) Hmo1 expressed in the human cell line HT1080 is mostly colocalized with UBF in nucleoli (upper panel). Hmo1 produced in the human cell line 3D-1 is concentrated in Pseudo-NORs with UBF1 (lower panel). (B) shRNA targeting both UBF1 and UBF2 in the human cell line HT1080 was used to mediate their depletion. Western blotting was used to assess depletion efficiency and Hmo1 expression after doxycycline induction. (C) Growth curves after induction of Hmo1 expression and depletion of UBF (UBF KD Hmo1+doxy) and after only UBF depletion (UBF KD+ doxy) or without doxycycline (UBF KD Hmo1) (UBFKD) show that Hmo1 cannot overcome the growth arrest associated with UBF depletion.
Figure 8.
Figure 8.
Pol I stimulation by Hmo1 requires the integrity of BoxA and the conserved motif CR. (A) Schematic representation of five truncated versions of Hmo1: BoxA, BoxAB, Hmo1ΔCR and Hmo1ΔBoxA. Hmo1 full size is represented on the top. Location of domains [BoxA (A), BoxB (B), CR, C terminal tail (C)] is indicated for each construct. (B) The five truncated versions of Hmo1, BoxA, BoxAB, Hmo1ΔCR, Hmo1ΔC and Hmo1ΔBoxA were YFP fused and expressed in an hmo1Δ background bearing nucleolar (mCherry-Nop1) and nuclear pore complex (CFp-Nup49) markers. Full size Hmo1 and BoxA were expressed in a WT strain bearing nucleolar (mCherry-Nop1) and nuclear pore complex (CFP-Nup49) markers. Selected cells are representative of the population. Scale bar = 5µm (C) The truncated Hmo1 constructions presented in the upper panel were functionally tested. Ten-fold serial dilutions of hmo1Δ-rpa49Δ (left) and hmo1Δ-rps23bΔ (right) strains expressing Hmo1 (Hmo1), or the four truncated constructions BoxA, BoxAB, Hmo1ΔC, Hmo1ΔCR or an empty vector (−) were spotted on plates containing 5-FOA to test for complementation. (D) The Hmo1 Box A can be functionally exchanged with the UBF1 dimerization motif. The BoxA of Hmo1 was swapped with the UBF1 dimerization motif to generate a chimeric protein Chim-Hmo1. Ten-fold serial dilutions of hmo1Δ-rpa49Δ (left) and hmo1Δ-rps23bΔ (right) strains expressing Hmo1 (Hmo1), Chim-Hmo1 or an empty vector (−) were spotted on plates containing 5-FOA to test for complementation.
Figure 9.
Figure 9.
Domain organization of Hmo1. Schematic representation of N-terminal part of UBF1, Hmo1 and Sp-Hmo1 containing a dimerization motif, a consensus HMG Box motif and a CR similar to the beginning of Box2 of UBF1. A part of the amino acid composition of these three domains is shown in the close-up view (dotted line; lower panel). Position of consensus HMGB is indicated, followed by the CR domain. Residues that are identical are in red.

Similar articles

See all similar articles

Cited by 21 articles

See all "Cited by" articles

References

    1. Lempiainen H, Shore D. Growth control and ribosome biogenesis. Curr. Opin. Cell. Biol. 2009;21:855–863. - PubMed
    1. Albert B, Perez-Fernandez J, Leger-Silvestre I, Gadal O. Regulation of ribosomal RNA production by RNA polymerase I: does elongation come first? Genet. Res. Int. 2012;2012:276948. - PMC - PubMed
    1. Merz K, Hondele M, Goetze H, Gmelch K, Stoeckl U, Griesenbeck J. Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules. Genes Dev. 2008;22:1190–1204. - PMC - PubMed
    1. O'Sullivan AC, Sullivan GJ, McStay B. UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat. Mol. Cell. Biol. 2002;22:657–668. - PMC - PubMed
    1. O'Mahony DJ, Rothblum LI. Identification of two forms of the RNA polymerase I transcription factor UBF. Proc. Natl. Acad. Sci. USA. 1991;88:3180–3184. - PMC - PubMed

Publication types

MeSH terms

Feedback