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. 2001 Nov;21(22):7731-46.
doi: 10.1128/MCB.21.22.7731-7746.2001.

A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins

T H King  1 , W A Decatur, E Bertrand, E S Maxwell, M J Fournier
Affiliations

A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins

T H King et al. Mol Cell Biol. 2001 Nov.

Abstract

Biogenesis of small nucleolar RNA-protein complexes (snoRNPs) consists of synthesis of the snoRNA and protein components, snoRNP assembly, and localization to the nucleolus. Recently, two nucleoplasmic proteins from mice were observed to bind to a model box C/D snoRNA in vitro, suggesting that they function at an early stage in snoRNP biogenesis. Both proteins have been described in other contexts. The proteins, called p50 and p55 in the snoRNA binding study, are highly conserved and related to each other. Both have Walker A and B motifs characteristic of ATP- and GTP-binding and nucleoside triphosphate-hydrolyzing domains, and the mammalian orthologs have DNA helicase activity in vitro. Here, we report that the Saccharomyces cerevisiae ortholog of p50 (Rvb2, Tih2p, and other names) is required for production of C/D snoRNAs in vivo and, surprisingly, H/ACA snoRNAs as well. Point mutations in the Walker A and B motifs cause temperature-sensitive or lethal growth phenotypes and severe defects in snoRNA accumulation. Notably, depletion of p50 (called Rvb2 in this study) also impairs localization of C/D and H/ACA core snoRNP proteins Nop1p and Gar1p, suggesting a defect(s) in snoRNP assembly or trafficking to the nucleolus. Findings from other studies link Rvb2 orthologs with chromatin remodeling and transcription. Taken together, the present results indicate that Rvb2 is involved in an early stage of snoRNP biogenesis and may play a role in coupling snoRNA synthesis with snoRNP assembly and localization.

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Figures

FIG. 1
FIG. 1
Kinetics of Rvb2 depletion in galactose-dependent test cells. Test cells dependent on a GAL::rvb2 allele (strain YWD313) were grown in medium containing 2% galactose, washed, and shifted to medium containing 2% glucose. Cell samples were collected before and at various intervals after the medium shift. (A) Northern analysis of RVB2 mRNA accumulation. Total RNA was extracted from cells at the time points indicated and fractionated on an agarose-formaldehyde gel, and a blot was probed with radiolabeled DNA that recognizes RVB2 mRNA. (B) Western analysis of Rvb2p protein accumulation. Total protein was extracted at the time points indicated, fractionated by SDS-polyacrylamide gel electrophoresis, and electroblotted to a nitrocellulose membrane. Proteins were detected with rabbit polyclonal IgG (raised against recombinant Rvb2p) and a goat anti-rabbit–peroxidase conjugate. Left lane, total protein from a strain (YWD312) carrying HIS3::rvb2 and Rvb2-GFP expressed from a plasmid; the other lanes show total proteins from GAL::rvb2 cells incubated in glucose for 0, 2, and 8 h, as indicated.
FIG. 2
FIG. 2
Rvb2p is required for accumulation of box C/D and H/ACA snoRNAs in yeast. Test cells dependent on a GAL::rvb2 allele (strain YWD313) were grown in galactose medium and shifted to glucose medium. Total RNA was extracted from cells harvested at various intervals following the shift, and snoRNAs were examined by Northern blot analysis. RNA loading was adjusted to obtain approximately equal amounts of tRNA per lane. The gel patterns show the effects of Rvb2p depletion on the accumulation of box C/D snoRNAs (A), box H/ACA snoRNAs (B), and RNase P, MRP, and 5S RNAs (C).
FIG. 3
FIG. 3
Accumulation of selected mRNAs in Rvb2p-depleted cells. GAL::rvb2 test cells were grown in medium containing 2% galactose, washed, and shifted to medium containing 2% glucose. Total RNA was extracted from cells collected before (left lane) and after (other lanes) the medium shift, fractionated on a 1.5% agarose-formaldehyde gel, and incubated with appropriate radiolabeled antisense probes. Incubation times in glucose are indicated above each lane. 18S rRNA served as a loading control.
FIG. 4
FIG. 4
Rvb2p is required for pre-rRNA processing. (A) Structure of the 35S pre-rRNA, showing the positions of cleavage sites and regions complementary to oligonucleotide probes 1 to 8. ITS, internal transcribed spacer. (B) Steady-state levels of pre-rRNA species in cells depleted of Rvb2 mRNA for 0 to 32 h. Total RNA was extracted from cells at the times indicated following shift from galactose to glucose and subjected to Northern analysis. Loading was adjusted to obtain approximately equal quantities of tRNA. The oligonucleotides used to identify the various rRNA species are as follows: probes 3 and 9, mature 18S and 25S rRNAs, respectively; probe 4, 20S and 35S precursors; probe 8, 27S and 7S precursors. (C) Mapping of the 5′ ends of the 35S precursor and products of the A1 and A2 cleavage reactions. The 5′ ends were identified by primer extension analysis using as the template total RNA from cells depleted of Rvb2 mRNA as described for panel B. Oligonucleotides 1, 3, and 5 were extended to the 5′ end of 35S rRNA and the A1 and A2 cleavage sites, respectively. (D) Pulse-chase analysis of pre-rRNA processing in GAL::rvb2 cells incubated in galactose (left) or glucose medium for 20 h (right). Cells were labeled with [3H]methionine for 2.5 min and chased with cold methionine. Samples were removed at the times indicated, and total RNA was extracted and fractionated on an agarose-formaldehyde gel.
FIG. 5
FIG. 5
Nuclear location of Rvb2p and mouse p50. (A) Distribution of Rvb2-GFP in yeast. Yeast cells (YWD312) each harboring a plasmid-borne copy of Rvb2-GFP (pWD235WGFP) were grown to log phase in selective medium. The cells were incubated with a Cy3-labeled oligonucleotide probe specific for U3, and the subnuclear localizations of p50-GFP and U3 were examined by fluorescence microscopy (top row). Nuclear DNA was visualized with DAPI (bottom left), and cell morphology was visualized with DIC (bottom right). (B) Localization of the mouse homolog of Rvb2 in COS-7 cells. Cells were transformed by the calcium-phosphate procedure (58) with a plasmid (pTK147) carrying mouse Rvb2 fused to GFP. Localization of Rvb2-GFP was observed by fluorescence microscopy (left), and nucleoli were visualized with phase contrast (right). Each field is 30 by 30 μm; arrows, nucleoli.
FIG. 6
FIG. 6
Loss of Rvb2p causes delocalization of C/D and H/ACA snoRNP proteins. GAL::rvb2 test strains containing chromosomally encoded Nop1p (YWD313) or a plasmid encoding a Gar1p-GFP fusion were grown in minimal selective galactose medium and shifted to glucose medium for 10 h to halt production of RVB2 mRNA (see Materials and Methods). Nop1p was detected with a primary monoclonal antibody, followed by a fluorescein isothiocyanate-coupled secondary antibody and observed by immunofluorescence microscopy. Gar1p-GFP was observed by direct fluorescence microscopy. (A) Localization of Gar1p in wild-type cells (upper left) and cells depleted of Rvb2p (lower left). (B) Localization of Nop1p in wild-type cells (upper left) and Rvb2p-depleted cells (lower left). The nucleus was visualized with a DNA stain (DAPI; middle). A merged view is also shown (right).
FIG. 7
FIG. 7
Point mutations in the Walker A and B domains disrupt growth. (A) Selected residues in Walker domains A and B of Rvb2p were substituted by site-directed mutagenesis. (B) Point mutations G80A and K81A (Walker A motif) cause temperature-sensitive growth defects. Left plate, permissive temperature (30°C); right plate, restrictive temperature (37°C). (C) The D296N allele is lethal and also toxic when overexpressed in a wild-type background. Growth of a wild-type strain (MH2-h) expressing a high-copy-number GAL1::rvb2 D296N allele is severely impaired in galactose medium (right, bottom two rows) but unaffected when expression is repressed by incubation in glucose (left, bottom two rows). No effects occur with the empty vector (left and right, top rows) or a vector containing a wild-type RVB2 allele under the control of the GAL1 promoter (left and right, second rows from top). The dilution series is indicated above each panel. N, no dilution.
FIG. 8
FIG. 8
Accumulation of snoRNA is impaired in cells harboring rvb2 ts mutations. Results of Northern blot analysis are shown for box H/ACA snoRNAs (A), box C/D snoRNAs (B), splicing snRNAs and RNase P (C), and EF-1β, ASC1, TEF4, RPS5, and RPL32 mRNAs (D). Panels A to C include total RNA from wild-type cells (WT) and cells with the rvb2 K81A allele (K81A) or G80A allele (G80A). Times of incubation at 37°C are indicated above each lane. Time zero identifies samples harvested prior to the temperature shift. Northern blots in panel D are of RNA extracted from cells grown at 30°C.
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