The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae  Martin Koš, David Tollervey  Molecular Cell  Volume 20, Issue 1, Pages 53-64 (October 2005) DOI: 10.1016/j.molcel.2005.08.022 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Pre-rRNA Processing Pathway in Saccharomyces cerevisiae Pre-rRNA cleavage and processing sites are indicated above the transcripts. Small numbered bars below the primary transcript represent oligonucleotides used as probes in Northern analyses. Modified from Venema and Tollervey (1999). Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Depletion of Dbp4p Leads to U14 Accumulation in High Molecular Weight Complexes (A) Growth of wild-type (wt) (CEN-PK2; D914) and GAL::3HA-dbp4 (D965) strains after glucose addition. Cells were pregrown in synthetic galactose media and were diluted with prewarmed medium to maintain exponential growth. Optical density (OD) at 600 nm was measured at the indicated times. The initial OD was normalized to 1. A regression line is shown for the wt strain. (B) Western blot analysis of the GAL::3HA-dbp4, PWP2-TAP (D964) strains after glucose addition. Samples were analyzed by Western blot analysis using an antibody against the HA tag on Dpb4p. Pwp2p-TAP is detected due to binding of the antibodies to the protein A tag. A nonspecific band migrating slightly below 3HA-Dbp4p is indicated by an asterisk. (C) Effect of Dbp4p and Rok1p depletion on the distribution of U14 on sucrose gradients. The DBP4, PWP2-TAP (D635), GAL::3HA-dbp4, PWP2-TAP (D964), and GAL::3HA-rok1, PWP2-TAP (D1030) strains were analyzed 3 hr after glucose addition. Cell lysates were fractionated by centrifugation through a 10%–50% sucrose gradient. RNA was isolated from each fraction and analyzed by Northern hybridization. Positions of the pre-RNA probes 003 and 004 are indicated in Figure 1. Fractions containing the 43S, 66S, and 90S preribosomal complexes are indicated. (D) Effects of Dbp4p depletion on the gradient distribution of snoRNAs involved in 18S rRNA maturation. Northern blots prepared as in (C) were hybridized with probes against all snoRNAs implicated in 18S rRNA modification or maturation. No signals were detected for snR52, snR53, snR55, snR56, or snR74. Arrows on the right indicate snoRNAs with altered sedimentation. Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 The Pools of Free snoRNPs Are Reduced in Rapidly Growing Cells (A) Wt (CEN-PK2, D914) and otherwise isogenic GAL::3HA-dbp4 (D965) strains were pregrown in minimal galactose medium containing ammonium sulfate as nitrogen source. Lysates were prepared and analyzed 3 hr after glucose addition as described for Figure 1C. (B) Wt CEN-PK2 and W303 strains and otherwise isogenic GAL::3HA-dbp4 strains were pregrown in minimal galactose medium containing proline as nitrogen source (SC-Gal-Pro). Lysates were prepared and analyzed 3 hr after glucose addition as described for Figure 1C, except that successive pairs of fractions were pooled. Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 U14 snoRNA Copurifies with Preribosomes Yeast strains D967 (PWP2::TAP), D968 (PWP2::TAP, GAL::3HA-dbp4), D969 (RIO2::TAP), and D970 (RIO2::TAP, GAL::3HA-dbp4) were grown in minimal galactose medium containing proline as nitrogen source (SC-Gal-Pro). Lysates were prepared 3 hr after glucose addition. Sepharose protein A was added to the lysates and incubated for 2 hr at 4°C. RNA was isolated from affinity-purified complexes and analyzed by Northern blotting using probes hybridizing to U14, snR41, and pre-rRNA probe 004 (see Figure 1). Minus signs indicate samples prepared after depletion of Dbp4p. An asterisk indicates a nonspecific signal resulting from a compression of the background by 25S rRNA. Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 U14 and snR41 snoRNAs Remain Base Paired to pre-rRNA in the Absence of Dbp4p (A) Wt (CEN-PK2, D914) and otherwise isogenic GAL::3HA-dbp4 (D965) strains were grown in minimal galactose medium containing proline as nitrogen source (SC-Gal-Pro). Lysates prepared 3 hr after glucose addition were treated with proteinase K for 3 hr at 14°C and fractionated in 10%–30% sucrose gradients. RNA was isolated from each fraction and analyzed by Northern blotting. The sedimentation profile of mature rRNAs from wt cells is shown as an ethidium bromide-stained agarose gel at the bottom. The pre-rRNAs were detected by hybridization with probe 004. (B) Coomassie-stained, SDS-polyacrylamide gel showing lysates before and after treatment with proteinase K. Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Characterization of Dbp4p Mutants (A) The GAL::3HA-dbp4 strain (D965) was transformed with plasmids expressing either wt Dbp4p or mutants in the motifs I and III under the control of the native promoter. Cells were grown into exponential phase in SC-Gal without tryptophan. 10-fold serial dilutions were spotted on plates containing glucose medium SC-Glu and grown for 2 days at 30°C. (B) ATPase assay: wt and mutant Dbp4p proteins were purified as GST fusions from E. coli. ∼1 μg of each fusion protein was incubated with or without total yeast RNA at 30°C, and the release of inorganic phosphate was measured by colorimetric assay. Samples were taken at indicated time points. (C) Yeast CEN-PK2 strain expressing either protein A-tagged wt or mutant Dbp4p from a GAL1 promoter on a centromeric plasmid were grown in SC-Gal media, lysed, and subjected to immunoprecipitation with IgG Sepharose. Total RNA was isolated from immunoprecipitates and analyzed by Northern blotting. (D–F) The GAL::3HA-dbp4 strain-expressing plasmid-borne wt Dbp4p (D976), the AAA mutant (D979), or GRT mutant (D1014) were pregrown in SC-Gal-Pro. Lysates were prepared and analyzed 3 hr after glucose addition, as described in Figure 1C. The pre-rRNAs (E) were detected by probes 004 and 020. The Western blot analysis (F) was performed by using an anti c-myc tag antibody, 9E-10 (Santa Cruz). Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Depletion of Dbp4p Abolishes A2 Cleavage in pre-rRNA Processing (A) RNA was extracted from the wt (CEN-PK2, D914) and otherwise isogenic GAL::3HA-dbp4 (D965) strains during growth in YPGal (0 hr samples) and at intervals after glucose addition. RNA was analyzed by Northern hybridization using probes directed against the pre-rRNAs and rRNAs. Probes are indicated on the left and RNAs on the right of the figure. See Figure 1 for locations of the probes and processing intermediates. (B) The same RNA as above was analyzed by Northern hybridization using probes against snoRNAs. Molecular Cell 2005 20, 53-64DOI: (10.1016/j.molcel.2005.08.022) Copyright © 2005 Elsevier Inc. Terms and Conditions