Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits - PubMed (original) (raw)

Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits

Joshua White et al. Mol Cell Biol. 2008 May.

Abstract

BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5' internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Delta mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Deletion of BUD23 results in reduced levels of 40S subunits. (A) Whole-cell extracts were prepared from wild-type (WT) and _bud23_Δ cells and fractionated by ultracentrifugation through 7 to 47% sucrose gradients. The positions of free 40S monosomes and free 60S and 80S monosomes are shaded. Note that in the _bud23_Δ sample, the largest peak corresponds to free 60S. (B) The ratio of small to large subunits was determined by making extracts in the absence of cycloheximide, to allow ribosome runoff, and in the absence of Mg2+ to dissociate 40S from 60S subunits. Extracts were fractionated as described above for panel A.

FIG. 2.

FIG. 2.

Northern blot analysis of pre-rRNA species in bud23 mutant cells. (A) Cartoon of the processing pathway of rRNA in yeast. The positions of oligonucleotide probes (probes a to h) used for Northern blotting are shown. 5′-ETS, external transcribed spacer. (B) Ten micrograms of total RNA prepared from wild-type (WT) (BY4741) and _bud23_Δ (AJY2161) cells was separated on a 1% agarose formaldehyde gel, transferred to a membrane, and probed with the indicated probes. (C) Cartoon depicting the indicated pre-rRNA species detected in panel B. (D) Pulse-chase labeling of rRNA was carried out as described in Materials and Methods. Wild-type (BY4741) and _bud23_Δ (AJY2161) (both containing pRS411, MET15) were labeled with [_methyl_-3H] methionine for 2 minutes and then chased with excess unlabeled methionine. Aliquots were collected and quickly frozen at the indicated times after labeling. Total RNA was separated on a formaldehyde agarose gel, transferred to a membrane, and exposed to X-ray film after spraying with En3Hance (Dupont). The positions of RNA species are identified to the right of the gel.

FIG. 3.

FIG. 3.

Rps2 and ITS1 accumulate in the nucleus in _bud23_Δ mutant cells. (A) Wild-type (WT) (BY4741) and _bud23_Δ cells (AJY2161) were transformed with pAJ1399 (_RPS2_-eGFP) and pAJ1629 (_SIK1_-mRFP). Fluorescence was visualized in green (GFP) and red (RFP) channels. The merged images and differential interference contrast (DIC) images are also shown. (B) Wild-type and _bud23_Δ cells were prepared for fluorescence in situ hybridization using a Cy3-labeled probe complementary to the 5′ end of ITS1. (C) For comparison, the localization of ITS1 was examined in AJY1539 [CRM1(T539C)] mutants cells not treated with LMB (−LMB) or treated with 0.1 μg/ml LMB (+LMB) for 30 min to block ribosomal subunit export.

FIG. 4.

FIG. 4.

Bud23 is highly conserved but restricted to eukaryotes. (A) BLAST sequence alignment of Bud23 orthologs. Bud23 orthologs were from the following species (the accession numbersof these orthologs shown in brackets): Saccharomyces cerevisiae (Scer) [6319895], Aspergillus nidulans (Anid) [67539238], Schizosaccharomyces pombe (Spom) [19115061], Arabidopsis thaliana (Athal) [15242087], Plasmodium falciparum (Pfal) [124506365], Drosophila melanogaster (Dmel) [21355093], Caenorhabditis elegans (Cele) [17552330], Strongylocentrotus purpuratus (Spur) [72006191]), Xenopus laevis (Xlae) [148234441], and Homo sapiens (Hsap) [23199995]. Gaps introduced to maximize alignment are indicated by dashes. (B) Phylogenetic tree of Bud23 orthologs calculated using the neighbor-joining method as described in Materials and Methods. Lengths of branches correspond to the estimated evolutionary distances between Bud23 variants from different taxons (the bar below the tree indicates the distance of 0.1 unit according to the JTT matrix). Numbers at the nodes indicate the statistical support for grouping of the corresponding taxons, calculated from 1,000 bootstrap resamplings of the alignment. Values close to 100 indicate strong support.

FIG. 5.

FIG. 5.

Structural model of Bud23. (A) Bud23 model shown in ribbon representation, colored according to the predicted accuracy (estimated agreement with the native structure). Ribbons are colored blue (highly confident, predicted error of ∼1 Å) to green (predicted low accuracy, expected differences between the model and the native structure of up to ∼5 Å) to red (predicted very low accuracy, error difficult to estimate). The orientation of two helices in the C terminus (right) with respect to each other and with respect to the catalytic domain (left) is completely arbitrary, as this region is likely to be disordered. (B) Bud23 model colored according to the distribution of electrostatic potential, from red (−3 kT) to blue (+3 kT).

FIG. 6.

FIG. 6.

Localization of mutant Bud23 proteins. (A) Wild-type (WT) and mutant Bud23 proteins were expressed as GFP fusions from plasmids in the _bud23_Δ mutant AJY2161. The constructs were as follows: WT (pAJ2151); Bud23ΔC, deleted of amino acids 220 to 275 (pAJ2160); Bud23ΔC(K4A), Bud23ΔC in which the lysines at positions 190, 191, 193 and 194 were changed to alanines (pAJ2164); Bud23ΔN, deleted of amino acids 1 to 219 (pAJ2175); and Bud23(K4A) (pAJ2176). Serial dilutions of cultures were spotted onto rich growth medium and incubated at 30°C for 3 days. (B) GFP fusions of wild-type and mutant Bud23 proteins, as in panel A, were expressed in a BUD23 wild-type strain that also expressed the nucleolar marker Sik1 as a fusion to RFP (19). Cells were fixed with formaldehyde before visualization in green (GFP) and red (RFP) channels. Differential interference contrast (DIC) images are shown for reference. The white arrowheads in the Bud23ΔC(K4A) GFP panel indicate the position of the nucleolus, where the GFP signal is diminished.

FIG. 7.

FIG. 7.

Bud23 methylates G1575 of 18S rRNA. (A) Secondary structure of the 3′ portion of yeast 18S rRNA (URL

http://www.rna.ccbb.utexas.edu/

). The black arrowhead indicates the position of G1575. (B) Structure of the 30S subunit from E. coli (PDB structure 29B0) (42) showing the position of G1338 (dark gray, filled), corresponding to G1575 in yeast and the position of the P-site tRNA (dark gray). (C) Total RNA was prepared from wild-type (WT) and _bud23_Δ mutant cells. Primer extension was carried out with a primer that would yield a 61-nucleotide product if extended to G1575. Products were resolved on a denaturing 8% polyacrylamide gel. A DNA sequence ladder using the same primer was run to identify the position of G1575.

FIG. 8.

FIG. 8.

Catalytically inactive bud23 mutants do not methylate G1575 but do complement the growth defect of a bud23 deletion. (A) Cartoon of Bud23 structure, showing mutated residues G57E and D77K that are part of the AdoMet binding pocket. The relative positions of the two β-strands and α-helix have been altered slightly from the structure modeled in Fig. 5. (B) Growth test of bud23 mutants. Cultures of a _bud23_Δ strain (AJY2161) carrying an empty vector (pRS315) (_bud23_Δ), plasmid-borne wild-type BUD23 (pAJ2154) (WT), and bud23(D77K) (pAJ2155) (D77K), and bud23(G57E) (pAJ2156) (G57E) mutants were standardized for cell density, and 10-fold serial dilutions were plated onto selective medium. Plates were incubated for 3 days at 30°C. (C) Total RNA was prepared from the strains in panel B. Primer extension was carried out as described in the legend to Fig. 7. The gray scale was adjusted in the three right lanes to equalize the signal for background primer extension stops.

FIG. 9.

FIG. 9.

Changing guanosine at position 1575 to adenosine has no discernible effect on cell growth. (A) Schematic of strategy to introduce mutant ribosomes into yeast. 5-Fluoroorotic acid (5FOA)-containing media selects against the URA3 vector, forcing cells to utilize the rDNA locus introduced on the LEU2 vector. WT, wild type. (B) LEU2 plasmids expressing wild-type rRNA (pAJ718) (WT), G1575A mutant (pAJ2157), or no rRNA (pRS425) (vector) were transformed into strain AJY1185 and tested for growth on 5FOA-containing medium. (C) A 2.7-kb fragment spanning the G1575A mutation of the plasmid-borne rDNA locus was amplified by PCR using primers AJO214 and AJO1051. The PCR product was treated with EcoRI and analyzed on an agarose gel. The brightest band of the 1-kb ladder is 3 kb.

References

    1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 253389-3402. - PMC - PubMed
    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1988. Current protocols in molecular biology. John Wiley & Sons, New York, NY.
    1. Bakin, A., and J. Ofengand. 1995. Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution. Nucleic Acids Res. 233290-3294. - PMC - PubMed
    1. Baranov, P. V., R. F. Gesteland, and J. F. Atkins. 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10221-230. - PMC - PubMed
    1. Bradatsch, B., J. Katahira, E. Kowalinski, G. Bange, W. Yao, T. Sekimoto, V. Baumgartel, G. Boese, J. Bassler, K. Wild, R. Peters, Y. Yoneda, I. Sinning, and E. Hurt. 2007. Arx1 functions as an unorthodox nuclear export receptor for the 60S preribosomal subunit. Mol. Cell 27767-779. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources