Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae - PubMed (original) (raw)

Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae

D Kressler et al. Mol Cell Biol. 1998 Apr.

Abstract

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

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Figures

FIG. 1

FIG. 1

Scheme of pre-rRNA processing in S. cerevisiae. (A) Structure and processing sites of the 35S precursor. This precursor contains the sequences for the mature 18S, 5.8S, and 25S rRNAs, which are separated by two internal transcribed spacers, ITS1 and ITS2. Two external transcribed spacers, 5′ ETS and 3′ ETS, are present at either end. The locations of the various probes (numbered from 1 to 9) used in this study are also indicated. Thick lines represent mature rRNA species, and thin lines represent transcribed spacers. (B) Pre-rRNA processing pathway. The 35S pre-rRNA is cleaved at site A0 by endonuclease Rnt1p (1), generating the 33S pre-rRNA. This molecule is subsequently processed at sites A1 and A2 to give rise to the 20S and 27SA2 precursors, resulting in the separation of the pre-rRNAs destined for the small and large ribosomal subunits. It is thought that the early pre-rRNA cleavages from A0 to A2 are carried out by a large small nucleolar RNP complex (67), which is likely to be assisted by the putative ATP-dependent RNA helicases Dbp4p (31), Fal1p (28), Rok1p (65), and Rrp3p (41). The final maturation of the 20S precursor takes place in the cytoplasm, where endonucleolytic cleavage at site D yields the mature 18S rRNA. The 27SA2 precursor is processed by two alternative pathways that both lead to the formation of mature 5.8S and 25S rRNAs. In the major pathway, the 27SA2 precursor is cleaved at site A3 by RNase MRP (67). The putative ATP-dependent RNA helicase Dbp3p (72) assists in this processing step. The resulting 27SA3 precursor is 5′-to-3′ exonucleolytically digested up to site B1S to yield the 27SBS precursor, a reaction requiring exonucleases Xrn1p and Rat1p (21). The minor pathway processes the 27SA2 molecule at site B1L, producing the 27SBL pre-rRNA. While processing at site B1 is completed, the 3′ end of mature 25S rRNA is generated by processing at site B2. The subsequent ITS2 processing of both 27SB species appears to be identical. Cleavage at sites C1 and C2 releases the mature 25S rRNA and the 7S pre-rRNA. The latter undergoes exosome-dependent 3′-to-5′ exonuclease digestion to the 3′ end of the mature 5.8S rRNA (36, 37); this reaction also requires the putative ATP-dependent RNA helicase Dob1p (12). The data presented in this study suggest that Dbp6p is required for the assembly of 60S ribosomal subunits, a process that may also involve three other putative ATP-dependent RNA helicases: Dbp7p (10), Drs1p (44), and Spb4p (49). See references and for reviews on pre-rRNA processing and _trans_-acting factors.

FIG. 2

FIG. 2

DBP6 encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family. Seven of the eight conserved motifs characteristic of DEAD-box proteins are found in Dbp6p (bold type and underlined). The helicase core region extends from amino acid 234 (A motif) to amino acid 598 (HRVGR motif). A portion of the N-terminal domain, starting at amino acid 39 and ending at amino acid 124, is highly enriched in serine and the negatively charged amino acids aspartic acid and glutamic acid (underlined).

FIG. 3

FIG. 3

Growth of yeast cells is impaired upon Dbp6p depletion. (A) Growth curves for YDK8-1A(pAS24-DBP6) (GAL::DBP6; open circles) and YDK8-1A(YCplac111-DBP6) (DBP6; closed circles) at 30°C after logarithmic cultures were shifted from YPGal medium to YPD medium for up to 36 h. Data are given as doubling times at different times in YPD medium. (B) Depletion of Dbp6p. Cell extracts of the GAL::DBP6 strain were prepared from samples harvested at the indicated times. The cell extract of the DBP6 strain was prepared from a sample harvested after 36 h in YPD medium. Extracts were assayed by Western blot analysis with monoclonal mouse anti-HA antibody 16B12. Equal amounts of protein (ca. 70 μg) were loaded in each lane, as judged by Coomassie blue staining of gels or red Ponceau staining of blots (data not shown). Prestained markers (Bio-Rad) were used as standards for molecular mass determinations. The HA-Dbp6p signal is indicated by an arrow. No signal was detected for untagged Dbp6p.

FIG. 4

FIG. 4

Depletion of Dbp6p results in a deficiency in 60S ribosomal subunits. YDK8-1A(pAS24-DBP6) was grown in YPGal medium and shifted to YPD medium for up to 24 h. Polysome analysis was done after 6 h (A) and 24 h (B). Cell extracts were resolved in 7 to 50% sucrose gradients. The peaks of free 40S and 60S ribosomal subunits, 80S ribosomes (free couples and monosomes), and polysomes are indicated. Half-mer polysomes are indicated by arrows.

FIG. 5

FIG. 5

HA-Dbp6p localizes to the nucleolus. Indirect immunofluorescence was performed with cells expressing HA-Dbp6p from the DBP6 promoter [YDK8-1A(pRS415-HA-DBP6)]. (A) HA-Dbp6p was detected with monoclonal mouse anti-HA antibody 16B12, followed by decoration with goat anti-mouse rhodamine-conjugated antibodies. (B) Nop1p was detected with polyclonal rabbit anti-Nop1p antibodies, followed by decoration with goat anti-rabbit fluorescein-conjugated antibodies. (C) Chromatin DNA was stained with DAPI.

FIG. 6

FIG. 6

Dbp6p depletion leads to reduced synthesis of the mature 25S and 5.8S rRNAs. (A) Wild-type control strain YDK8-1A(YCplac111-DBP6) (DBP6) and strain YDK8-1A(pAS24-DBP6) (GAL::DBP6) were grown at 30°C in YPGal medium, shifted for 12 h to YPD medium, and then grown for 10 h in SD-Met. Cells were pulse-labeled (p) for 1 min with [_methyl_-3H]methionine and then chased (c) for 2, 5, and 15 min with an excess of unlabeled methionine. Total RNA was extracted, and 20,000 cpm was loaded and separated on a 1.2% agarose–formaldehyde gel, transferred to a nylon membrane, and visualized by fluorography. (B) Strains YDK8-1A(pRS416-DBP6) (DBP6) and YDK8-1A(pAS24-DBP6)(pRS416) (GAL::DBP6) were grown at 30°C in SGal-Ura and then shifted to SD-Ura for 22 h. Cells were pulse-labeled (p) for 2 min with [5,6-3H]uracil and then chased (c) for 5, 15, 30, and 60 min with an excess of unlabeled uracil. Total RNA was extracted, and 30,000 cpm was loaded and separated on a 7% polyacrylamide–8 M urea gel, transferred to a nylon membrane, and visualized by fluorography. The positions of the different pre-rRNAs, mature rRNAs, and tRNAs are indicated.

FIG. 7

FIG. 7

Dbp6p depletion leads to lower steady-state levels of the 27S precursors and the mature 25S rRNA. Strains YDK8-1A(pRS416-DBP6) (DBP6) and YDK8-1A(pAS24-DBP6) (GAL::DBP6) were grown in YPGal medium and shifted to YPD medium for up to 36 h. The cells were harvested at the indicated times. Total RNA was extracted, separated on a 1.2% agarose–formaldehyde gel, transferred to a nylon membrane, and subjected to Northern analysis. The same filter was consecutively hybridized with the probes indicated in Fig. 1A. (A) Oligonucleotides 2 and 9, base pairing to sequences within the mature 18S and 25S rRNAs, respectively. (B) Oligonucleotide 1 in 5′ ETS. (C) Oligonucleotide 3 in ITS1 between sites D and A2. (D) Oligonucleotide 4 in ITS1 between sites A2 and A3. (E) Oligonucleotide 5 in ITS1 downstream of site A3. (F) Oligonucleotide 7 in ITS2 between sites E and C2. The positions of the different pre-rRNAs and mature rRNAs are indicated.

FIG. 8

FIG. 8

Dbp6p depletion leads to lower steady-state levels of the 7S precursor and the mature 5.8S rRNAs. Strains YDK8-1A(pRS416-DBP6) (DBP6) and YDK8-1A(pAS24-DBP6) (GAL::DBP6) were grown in YPGal medium and shifted to YPD medium for up to 36 h. The cells were harvested at the indicated times. Total RNA was extracted, separated on a 7% polyacrylamide–8 M urea gel, transferred to a nylon membrane, and subjected to Northern analysis. The same filter was consecutively hybridized with three different probes: oligonucleotide 7 in ITS2 between sites E and C2 (top panel); oligonucleotide 6, base pairing to sequences within the mature 5.8S rRNAs (middle panel); and oligonucleotide 5S, base pairing to sequences within the mature 5S rRNA (bottom panel). The positions of the 7S pre-rRNA and the mature 5.8S and 5S rRNAs are indicated.

FIG. 9

FIG. 9

Primer extension analysis of the 27S precursors and the mature 5.8S rRNAs. Strains YDK8-1A(pRS416-DBP6) (DBP6) and YDK8-1A(pAS24-DBP6) (GAL::DBP6) were grown in YPGal medium and shifted to YPD medium for up to 36 h. The cells were harvested at the indicated times, and total RNA was extracted. (A) Primer extension with oligonucleotide 7 in ITS2 reveals processing sites B1S, B1L, A3, and A2. The bottom panel is a longer exposure of the same gel, and it shows the A3 site. (B) Primer extension with oligonucleotide 6, priming within the mature 5.8S rRNAs. The positions of the primer extension stops corresponding to the different pre-rRNA species and the mature 5.8S rRNAs are indicated.

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