The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast.

Abstract

The m~A1779m~A178, dimethylation at the 3' end of the small subunit rRNA has been conserved in evolution from bacteria to eukaryotes. The yeast 18s rRNA dimethylase gene DIM1 was cloned previously by complementation in Escherichia coli and shown to be essential for viability in yeast. A conditional GAL1O::diml strain was constructed to allow the depletion of Dimlp from the cell. During depletion, dimethylation of the pre-rRNA is progressively inhibited and pre-rRNA processing at cleavage sites A1 and A2 is concomitantly lost. In consequence, the mature 18s rRNA and its 20s precursor drastically underaccumulate. This has the effect of preventing the synthesis of nonmethylated rRNA. To test whether the processing defect is a consequence of the absence of the dimethylated nucleotides or of the Dimlp dimethylase itself, a cis-acting mutation was created in which both dimethylated adenosines are replaced by guanosine residues. Methylation cannot occur on this mutant pre-rRNA, but no clear pre-rRNA processing defect is seen. Moreover, methylation of the wild-type pre-rRNA predominantly occurs after cleavage at sites A1 and A2. This shows that formation of the m~A , , , , r n~~, ,~, dimethylation is not required for pre-rRNA processing. We propose that the binding of Dimlp to the pre-ribosomal particle is monitored to ensure that only dimethylated pre-rRNA molecules are processed to 18s rRNA.

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…RNA isolation, Northern hybridizations, and primer extensions were carried out as previously described (Tollervey, 1987;Beltrame & Tollervey, 1992;Lafontaine et al+, 1995;Venema & Tollervey, 1996)+ 39 end-labeling of RNAs was performed as described by Ganot et al+ (1997b)+ Northern hybridizations and primer extensions designed to analyze pre-rRNA processing defects were carried out using the following oligonucleotides (see Fig+ 6A for the hybridization positions of these oligos): 59-CATGGCTTAATCTTTGAGAC-39 (probe 1); 59-CTCACGACGGTCTAAACCC-39 (probe 2); 59-CACCCATTCCCTCTTGCTAG-39 (probe 3); 59-TTAAG CGCAGGCCCGGCTGG-39 (probe 4); 59-GATTGCTCGA ATGCCCAAAG-39 (probe 5); and 59-GGCCAGCAATTT CAAGTTA-39 (probe 6)+ Antisense oligonucleotide probes used FIGURE 11. Northern analysis of snoRNA steady-state levels in cells lacking Rrp8p+ RNAs were extracted from the rrp8⌬ (JG544, lanes 2 and 4) and isogenic wild-type strain (JG540, lanes 1 and 3) grown at 30 8C (lanes 1 and 2) or 19 8C (lanes 3 and 4), separated by PAGE on an 8% polyacrylamide gel and transferred on a nylon membrane+ Indicated snoRNAs were detected using specific antisense oligodeoxynucleotide probes+ to detect various H/ACA and C/D snoRNAs are listed in Henras et al+ (1998)+ Pulse-chase labeling experiments were performed as follows+ Cells were grown to OD ; 0+4 in minimal medium either at 19 or 30 8C+ To 4 mL (30 8C) or 6 mL (19 8C) samples, 300 mCi (methyl-3 H)methionine were added+ After 2 min (30 8C) or 4 min (19 8C) labeling, 0+4 mL of 0+1 M methionine were added, and 1-mL cell samples were collected at 1, 2, 5, and 10 min (30 8C) or at 2, 5, 10, 15, and 20 min (19 8C) following chase+ Sedimentation of a total cellular extract on glycerol density gradient A total cellular extract was prepared as follows from strain JG590 expressing ZZ-Rrp8p+ Five hundred milliliters of culture at 1+2 ϫ 10 7 cells/mL were collected, washed with sterile water, and resuspended in 5 mL of A200 buffer [200 mM K-acetate, 20 mM Tris-Cl, pH 8+0, 5 mM Mgacetate, 0+2% Triton X-100, 1 mM dithiothreitol, and protease inhibitors benzamidine (2 mM), Leupeptine (0+5 mg/ mL), Pepstatin A (1 mg/mL), Chymostatin (2 mg/mL), Aprotinin (1 mg/mL)]+ Cells were lyzed in a "One shot cell disrupter" (Cell8D, Constant Systems Ltd+) at 1+7 kBar+ The extract was clarified by centrifugation for 15 min at 10,000 ϫ g and the total protein concentration in the supernatant was estimated using the Bio-Rad assay (Hercules, California)+ Five milligrams of total proteins were loaded in a 500-mL sample onto a 10-30% (v/v) glycerol gradient containing 20 mM HEPES-KOH, pH 7+9, 60 mM KCl, 1 mM MgCl 2 and protease inhibitors+ The gradient was centrifuged 10 h at 25,000 rpm in a SW41 Ti rotor (Beckman, Palo Alto, California) and 500-mL fractions were collected+ Proteins were precipitated using trichloro-acetic acid and separated by SDS-PAGE+…”

The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs

“…The three other eukaryotic positive ORFs with very long N-terminal extensions, in human, C. elegans, and A. thaliana, do not necessarily represent orthologs of Trm3p+ Intriguingly, the human ORF encodes a nuclear protein that binds with both high affinity and marked specificity to TAR RNA of HIV, hence its name of TRP-185 for 185-kDa TAR RNA protein (Sheline et al+, 1991;Wu et al+, 1991)+ Its binding is strongly dependent on the TAR RNA loop sequences and is stimulated by a set of cellular factors that also stimulate the binding of RNA polymerase II to HIV TAR RNA+ Binding of TRP-185 and RNA polymerase II to TAR RNA is mutually exclusive and TRP-185 stimulates gene expression from the HIV LTR in vitro (Wu-Baer et al+, 1995a, 1995b, 1996+ Interestingly, defective assembly of large ribosomal subunits in Pet56 mutants seems caused by inactivation of the Pet56 protein rather than by the lack of 21S mitochondrial rRNA G2270 methylation normally catalyzed by this protein, suggesting an additional role for the Pet56 methylase, possibly as a rRNA chaperone (Mason et al+, 1996)+ Likewise, although the presence of Dim1p, which catalyzes formation of the m 2 6 m 2 6 A doublet at the 39 end of 18S rRNA, is a critical factor for pre-rRNA processing, the absence of rRNA base-dimethylation itself does not result in any clear rRNA processing defect (Lafontaine et al+, 1995)+ Moreover, the gene for tRNA m 5 U54-methyltransferase is essential for viability in E. coli, although the known catalytic activity of the methylase is not (Persson et al+, 1992), and the vaccinia virus VP39 29-O-ribose methylase is a bifunctional protein (Shi et al+, 1996), like several other polypeptides (Smith, 1994;Henikoff, 1987)+ In this context, the presence of a long N-terminal extension in the yeast tRNA (Gm18) methylase is consistent with Trm3p having a more complex role than that of a mere RNA modifying enzyme+…”

The box H+ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase

“…Nine micrograms of total RNA was used for the Northern and primer-extension experiments presented in Figures 2, 3, 5 and 6, whereas 4.5 µg was used for the Northern analysis presented in Figures 4 and 9. Oligonucleotides used for pre-rRNA hybridization were oligonucleotides a, b, c, d, g, h described previously by Lafontaine et al (1995) as oligonucleotides d, g, h, i, k, l, respectively, and oligonucleotide f described previously as oligonucleotide b by Mitchell et al (1996). Oligonucleotides anti-U3, U14, MRP, snR10, and snR190 were as described previously (Girard et al 1992;Dichtl and Tollervey 1997 Sharma et al (1996).…”

“…During growth, cells were diluted with prewarmed medium and constantly maintained in exponential phase. RNA extraction, Northern hybridization, and primer extension were as described by Lafontaine et al (1995). Standard 1.2% agarose/formaldehyde and 8% acrylamide gels were used to analyze the processing of the high and low molecular weight rRNAs species, respectively (Tollervey 1987).…”

“…Formation of ⌿ residues is thought to occur through base rotation about the C 3 -C 6 axis after cleavage of the glycosyl bond (Goldwasser and Heinrikson 1966; for review, see Ofengand et al 1995). Additionally, a few positions are modified at the base level; the best described example being the universally conserved m 6 2 Am 6 2 A doublet at the 3Ј-end of the 18S rRNA (Lafontaine et al 1995). Recent data have shown that, in eukaryotes, the sites of both 2Ј-O-methylation (Cavaillé et al 1996;Kiss-Lá szló et al 1996;Nicoloso et al 1996) and ⌿ formation (Ganot et al 1997a;Ni et al 1997) in the rRNAs are selected by site-specific base-pairing of small nucleolar RNAs (snoRNAs) to the pre-rRNAs (for review, see Maden 1996;Tollervey 1996;Bachellerie and Cavaillé 1997;Smith and Steitz 1997).…”