Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase - PubMed (original) (raw)

Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase

Markus T Bohnsack et al. EMBO Rep. 2008 Dec.

Abstract

In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA--U3, U14 and snR30--whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

A quantitative screen to analyse the levels of all yeast snoRNAs. (A) Scheme to illustrate sample preparation and analysis (adapted from Ro et al, 2006; see main text and Methods for details). (B) Levels of free and pre-ribosome-bound snoRNAs were analysed on depletion of eight putative RNA helicases required for the biogenesis of the small ribosomal subunit. Soluble material from lysates of wild-type and depleted cells was centrifuged in sucrose gradients, and fractions containing either free or pre-ribosome-bound snoRNAs were pooled (see Fig 3). RNA was extracted and analysed as shown in (A). Data from depletions were normalized to wild-type samples processed in parallel. Average ratios of pre-ribosome bound to unbound snoRNAs in the indicated depletion strains shown were calculated from at least three independent experiments. Error bars represent standard error. U14 accumulates on pre-ribosomes on depletion of Dbp4 (green bar) and Has1 (red); a minor accumulation is observed for depletion of Dbp8 (blue). P tet, tetracycline-repressible promoter; snoRNAs, small nucleolar RNAs.

Figure 2

Figure 2

Depletion of the putative RNA helicase Rok1 leads to accumulation of snR30 on pre-ribosomes. Rok1 and Dhr2 were depleted and the levels of free and pre-ribosome-bound pools of all yeast snoRNAs were analysed by using qPCR. Data are presented as described for Fig 1B. On depletion of Rok1, but not Dhr2, snR30 accumulates on pre-ribosomes. P tet, tetracycline-repressible promoter; qPCR, quantitative PCR; snoRNAs, small nucleolar RNAs.

Figure 3

Figure 3

snR30 is retained on pre-ribosomes following the depletion of Rok1. Northern blot analysis of sucrose gradient fractions loaded with soluble material from either wild-type (WT) cells or after helicase depletion. Strains were grown in a medium containing doxycycline for helicase depletion. After lysis, soluble material was fractionated on sucrose gradients, RNA was isolated and analysed by Northern blot for the distribution of snR30. In the WT, as well as Has1 and Dbp4 depletion strains, snR30 is present largely in the top fractions of the gradient, whereas it shifts into higher molecular weight complexes on depletion of Rok1. Pools are indicated as the pre-ribosomal fractions (pool 2) and the gradient fractions containing the largest unbound fraction across all snoRNAs (pool 1). Pools were defined by comparison to the gel, which was stained before Northern transfer. Pool 1 contains material sedimenting well above the 40S ribosomal subunits. Pool 2 contains 40S and 60S ribosomes, together with the 90S pre-ribosomes. The 40S and 60S peaks were identified by ethidium staining of the gel. The 90S peak was identified by hybridization of an identical gradient with a probe directed against the ITS1 region of the 35S pre-rRNA. P tet, tetracycline-repressible promoter.

Figure 4

Figure 4

snR30 shows increased association with high molecular weight RNA following the depletion of Rok1. Northern blot analysis of 10–30% sucrose gradient fractions loaded with deproteinized RNA from P tet -rok1 cells expressing Rok1 (−Dox samples) or after helicase depletion (+Dox samples). For deproteinization, lysates were treated with proteinase K in a buffer containing 1% lithium dodecyl sulphate for 3 h at 4°C, before loading on sucrose gradients. To dissociate RNA–RNA interactions, +Dox samples were, in addition, incubated at 65°C for 3 min before loading the sucrose gradient. Dox, doxycycline; P tet, tetracycline-repressible promoter.

Figure 5

Figure 5

Analysis of Rok1 mutants for growth complementation and snR30 distribution. (A–C) Growth complementation analysis of Rok1 with mutations in motifs I (rok1-m1) and III (rok1-m3). In a background in which the genomic ROK1 gene is under control of the tetracycline-repressible promoter (P tet -rok1), growth complementation was tested with plasmid-derived wild-type (WT), mutant Rok1 or empty plasmid (vector). Dilutions of cultures were spotted on plates (A) without doxycycline or (B) under P tet -rok1 depletion conditions on plates containing doxycycline. Growth of the strains was also analysed in liquid culture (C). Although the rok1-m3 mutant can complement for growth, the mutation in motif I (rok1-m1) results in a slight dominant-negative effect. (D) Analysis of snoRNA accumulation on pre-ribosomes in the Rok1 complementation strains described in (A–C) shown in black bars, the P tet -rok1 strain (grey) and wild type in white (bars 1–6 for each snoRNA). The complementation strains are shown in the same order as in (A–C). Average ratios of pre-ribosome-bound to unbound snoRNAs from two separate experiments are shown. Although plasmid-derived wild-type Rok1 and rok1-m3 can largely rescue the phenotype of snR30 accumulation, the empty vector cannot; the rok1-m1 mutant shows an intermediate phenotype. OD, optical density.

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