Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

Abstract

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0). Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

Figure 1

Pre-rRNA processing in S.cerevisiae. (A) Structure and processing sites of the 35S pre-rRNA. This precursor contains the sequences for the mature 18S, 5.8S and 25S rRNAs, which are separated by the two internal transcribed spacers ITS1 and ITS2 and flanked by the two external spacers 5′-ETS and 3′-ETS. The location of the various probes (labeled B–I) used in this study are indicated. (B) Pre-rRNA processing pathway. The 35S pre-rRNA is cleaved at site A₀ by the endonuclease Rnt1p 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 separation of the pre-rRNAs destined for the small and large ribosomal subunits. It is thought that the early pre-rRNA cleavages A₀–A₂ are carried out by a large snoRNP complex, which is likely to be assisted by the putative ATP-dependent RNA helicases Dbp4p, Fal1p, Rok1p, Rrp3p, Dhr1p, Dhr2p and Dbp8p. Final maturation of the 20S precursor takes place in the cytoplasm, where an 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. The putative ATP-dependent RNA helicase Dbp3p assists in this processing step. The 27SA₃ precursor is 5′→3′ exonucleolytically digested up to site B_1S to yield the 27SB_S precursor, a reaction requiring the exonucleases Xrn1p and Rat1p. A minor pathway processes the 27SA₂ molecule at site B_1L, producing the 27SB_L 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′→5′ exonuclease digestion to the 3′-end of the mature 5.8S rRNA; this reaction also requires the putative ATP-dependent RNA helicase Dob1p. Assembly of the 60S subunit and the associated pre-rRNA processing reactions requires four other putative ATP-dependent RNA helicases: Dbp6p, Dbp7p, Dbp10p and Spb4p. The roles of the Drs1p and Mak5p proteins have not yet been defined. Only trans-acting factors belonging to the class of putative RNA helicases are indicated. For reviews on pre-rRNA processing and trans-acting factors see Kressler et al. (28) and Venema and Tollervey (29).

Figure 2

Alignment of the yeast and human Dbp8p proteins. Identical residues are marked * below the sequence. The eight highly conserved motifs are indicated by a bar in between the sequences. It is important to note that identical residues are found throughout the two proteins including the N- and C-terminal extension of the helicase core domain. The last 15 residues of the human protein are not shown. The alignment was performed using ClustalW at EBI (www.ebi.ac).

Figure 3

Depletion of Dbp8p impairs growth of yeast cells. (A) Growth comparison of MCD8H-3A (wt), MCD8H-3C [pMCD8-1] (DBP8) and MCD8H-3C [pMCD8-2] (GAL::DBP8) strains. Freshly grown cells were streaked on YPGal (galactose) and YPD (glucose) plates and incubated at 30°C for 3 days. (B) Growth curves of MCD8H-3C [pMCD8-1] (DBP8; closed circles) and MCD8-3H [pMCD8-2] (GAL::DBP8; open circles) at 30°C after logarithmic cultures were shifted from YPGal to YPD medium for up to 36 h. Data are given as estimated doubling time values at different time points in YPD. (C) Western blot analysis of depletion of Dbp8p. Whole cell lysates of the GAL::DBP8 strain were prepared from samples harvested at the indicated times. Equal amounts of proteins were separated by 12.5% SDS–PAGE and HA–Dbp8p (indicated by an arrow) was detected by western blot using monoclonal mouse anti-HA antibody 16B12 and goat anti-mouse alkaline phosphatase-conjugated antibodies. Prestained broad range molecular weight markers (Bio-Rad) were used as standards for molecular mass determination.

Figure 4

Depletion of Dbp8p results in a deficit in 40S ribosomal subunits and a decrease in polysomes. The DBP8 strain MCD8H-3C [pMCD8-1] (A) was grown in YPD at 30°C. The GAL::DBP8 strain MCD8H-3C [pMCD8-2] was grown at 30°C in YPGal and shifted to YPD for 6 (B), 12 (C) and 18 h (D). Cells were harvested at an OD₆₀₀ of 0.8 and similar amounts of cell extracts (12 A₂₆₀ units) were resolved in 7–50% (w/v) sucrose gradients. Gradient analysis was performed with an ISCO UV-6 gradient collector with continuous monitoring at A₂₅₄. Sedimentation is from left to right. The peaks of free 40S and 60S ribosomal subunits, 80S free couples/monosomes and polysomes are indicated. Due to a strong increase in the free 60S ribosomal subunit, the corresponding peak overlaps the free 80S/monosome peak in (D).

Figure 5

Synthesis of the mature 18S rRNA is strongly reduced in Dbp8p-depleted cells. Strains MCD8H-3C [pMCD8-1] (DBP8) and MCD8H-3C [pMCD8-2, YCplac33] (GAL::DBP8) were grown at 30°C in SGal-Ura then shifted to SD-Ura for 20 h. Cells were pulse labeled with [5,6-³H]uracil for 2 min and then chased with an excess of cold uracil. Total RNA was extracted from cell samples harvested at the indicated chase time points, resolved on a 1.2% agarose–6% formaldehyde gel, transferred to nylon membrane and visualized by fluorography. Approximately 25 000 c.p.m. was loaded in each lane. The positions of the different pre-rRNAs and mature rRNAs are indicated.

Figure 6

Depletion of Dbp8p affects the steady-state levels of pre-rRNA and mature rRNA species. Strains MCD8H-3C [pMCD8-1] (DBP8) and MCD8H-3C [pMCD8-2] (GAL::DBP8) were grown in YPGal then shifted to YPD. Cells were harvested at the indicated times and total RNAs were extracted. Equal amounts (5 µg) of total RNA were resolved on a 1.2% agarose–6% formaldehyde gel and transferred to a nylon membrane. The same membrane was stained with methylene blue (A) and then consecutively hybridized with the different probes B–F indicated in Figure 1A (B–F, respectively). The positions of the different pre-rRNAs and mature rRNAs are indicated.

Figure 7

Primer extension analyses of the 27S precursors and the 33S pre-rRNA. The same samples of total RNA analyzed by northern blot in Figure 6 were used for primer extension analyses. (A) Primer extension through 5′-ETS was performed using oligonucleotide I and allows detection of the 33S pre-rRNA (site A₀). (B) Primer extension using oligonucleotide E in ITS1 allows detection of the 27SA₃ pre-rRNA (site A₃). (C) Primer extension using oligonucleotide G in ITS2 reveals processing sites A₂, B_1L and B_1S, allowing detection of 27SA₂, 27SB_L and 27SB_L pre-rRNA species, respectively. The elevated signals in lane 8 (wt 36 h) are due to erroneously elevated RNA levels loaded on the gel.

Figure 8

HA-Dbp8p localizes to the nucleolus. Indirect immunofluorescence was performed with cells expressing HA–Dbp8p from the DBP8 promoter (MCD8-3C [pMCD8-6]). (A) DNA was stained with DAPI. (B) The nucleolar protein Nop1 was detected with polyclonal rabbit anti-Nop1p antibodies, followed by decoration with goat anti-rabbit rhodamine-conjugated antibodies. (C) HA–Dbp8p was detected with monoclonal mouse anti-HA antibody 16B12, followed by decoration with goat anti-mouse fluorescein-conjugated antibodies. (D) Superimposition of the signals obtained from Nop1p and HA–Dbp8p.

Figure 9

Site-directed mutagenesis of Dbp8p. (A) Four of the RNA helicase conserved motifs were individually altered by site-directed mutagenesis. The name of the mutant allele, the nature of the mutation and the associated growth phenotype are indicated. (B) Polysome analysis of MCD8-3C [pMCD8-11A] (dbp8-3) grown in YPD at 30°C. Twelve A₂₆₀ units of cellular extract were resolved on a 7–50% sucrose gradient. Sedimentation is from left to right. The peaks of free 40S and 60S ribosomal subunits, 80S free couples/monosomes and polysomes are indicated. (C) Western blot analysis of the mutant proteins. Equal amounts of total protein extracts from MCD8H-3C [pMCD8-1] carrying, respectively, YCplac111 (lane 1), pMCD8-8 (ProtA–Dbp8p; lane 2), pMCD8-9B (ProtA–Dbp8-1; lane 3), pMCD9-10B (ProtA–Dbp8-2; lane 4), pMCD8-11B (ProtA–Dbp8-3; lane 5) and pMCD8-12A (ProtA–Dbp8-4; lane 6) were separated by 10% SDS–PAGE. The ProtA-tagged proteins were detected by western blot using rabbit immune serum and goat anti-rabbit alkaline phosphatase-conjugated antibodies. Pre-stained low range standard (Bio-Rad) was used as the standard for molecular mass determination.