Comprehensive mutational analysis of yeast DEXD/H box RNA helicases involved in large ribosomal subunit biogenesis

Abstract

DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.

FIG. 1.

(A) Conserved motifs in DEXH/D box RNA helicases. Motifs I and II (DEXD/H box) are required for ATP binding and hydrolysis, whereas motifs III (SAT) and VI are required for RNA unwinding and RNP remodeling functions. The mutations that we made are shown. (B) Schematic of pre-rRNA processing in yeast. Pre-rRNA processing of the 18S, 5.8S, and 25S rRNAs begins on the 35S pre-rRNA primary transcript. Cleavage at A₂ in internal transcribed spacer 1 (ITS1) in the 35S pre-rRNA separates the 18S rRNA (which is incorporated into the SSU) from the 5.8S and 25S rRNAs (which are incorporated into the LSU). Processing of the 5.8S and 25S rRNAs occur through two distinct pathways. The main difference is that in the major pathway, the 5′ end of 5.8S is cleaved at B1S, whereas in the minor pathway the 5′ end of 5.8S is extended to B1L (the long form). ETS, external transcribed spacer.

FIG. 2.

Comparison of the protein levels of genomically tagged RNA helicases to the levels of wild-type and mutated RNA helicases expressed from plasmids. TAP-tagged RNA helicases created by genomic integration and yeast transformed with wild-type or mutated TAP-tagged RNA helicase plasmids (pGAD3 or pYES2) were grown to early log phase. Yeasts transformed with the helicase plasmids were shifted into SG-URA medium for 5 h. Protein was extracted, and equal amounts were run on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and blotted with PAP to detect the TAP tag. The blot was reprobed with anti-Mpp10 rabbit antibodies.

FIG. 3.

Screen by serial dilution on plates for dominant negative growth defects in yeasts overexpressing mutant RNA helicases. The yeast parent strain, YPH499, was transformed with plasmids containing wild-type or mutated RNA helicases, as indicated. Yeasts were grown to early log phase, serial diluted (1, 10, 100, and 1,000 times) onto GAL or DEX plates, and incubated at 17°C, 23°C, or 30°C. Gene expression from the plasmid bearing the helicase was observed when plating on GAL was carried out.

FIG. 4.

Growth curves of yeast overexpressing mutant RNA helicases. The yeast parent strain, YPH499, was transformed with plasmids containing wild-type or mutated RNA helicases. Yeasts were grown to early log phase at 30°C in DEX medium and then shifted into GAL medium for 30 h. Growth was recorded with a spectrophotometer at 0, 6, 12, 24, and 30 h of expression in GAL medium.

FIG. 5.

Analysis of pre-rRNA processing in yeast expressing wild-type or mutant RNA helicase plasmids. Yeasts were grown in DEX medium until early log phase (0 h of expression) and then shifted into GAL medium for 12 or 24 h (12 or 24 h of expression). RNA was extracted, run on a 12.5% agarose-formaldehyde gel, and transferred to a Hybond N+ membrane. The membrane was then probed with oligonucleotide C (which detects 35S, 32S, 27SA, and 23S pre-rRNAs), oligonucleotides B and E (which detect 35S, 27SA₂, 27SB, 23S, and 20S pre-rRNAs), and oligonucleotides A and Y (which detect 18S and 25S rRNAs). RNAs extracted from strains expressing Mtr4 and Spb4 plasmids were also run on 10% polyacrylamide gels and transferred to a Hybond N+ membrane. These membranes were probed with oligonucleotides to detect 7S pre-rRNA, 5.8S rRNA, and 5S rRNA.

FIG. 6.

The function of the mutant RNA helicases in vivo. The ability of the wild-type or mutant RNA helicase expressed from plasmids to restore growth when the endogenous helicase is depleted was assayed by serial dilution. Wild-type or mutant RNA helicase plasmids were transformed into yeast strains containing each respective helicase under a TET-repressible promoter. Yeasts were grown to early log phase in DEX medium, serially diluted (1, 10, 100, and 1,000 times) onto GAL plates with doxycycline (GAL + DOX), and incubated at 17°C, 23°C, or 30°C.

FIG. 7.

Some RNA helicases coimmunoprecipitate other proteins required for ribosome biogenesis or other helicases. Protein extracts were made from the indicated yeast strains and immunoprecipitated using anti-HA antibodies conjugated to beads. Immunoprecipitated protein (IP) and total protein extracted (T; 5% of total extract) were run on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were Western blotted with PAP antibodies which detect the protein A part of the TAP tag (anti-TAP), anti-Mpp10 antibodies (anti-Mpp10) which detects a protein component of the SSU processome, and anti-HA antibodies (anti-HA) which detect the immunoprecipitated protein. BA, beads alone. (A) SSU processome proteins (Utp7, Utp8, Utp9, and Utp10) immunoprecipitate Has1; proteins required for LSU biogenesis (Dbp10 and Mak5) immunoprecipitate Has1. (B) Mak5 is immunoprecipitated by other RNA helicases required for LSU biogenesis (Dpb3, Dpb6, Dbp9, Dbp10, and Mtr4) but not other nonribosomal proteins required for large subunit biogenesis (Rpf1 and Rpf2). (C) Dbp10, Spb4, and Mtr4, three RNA helicases required for synthesis of the 5.8S rRNA, coimmunoprecipitate each other but not RNA helicases required for early steps in pre-LSU-rRNA processing (Dbp3 and Drs1). (D) When nonribosomal proteins required for LSU biogenesis (Rpf2 and Rpf1) are depleted, Dbp10 no longer immunoprecipitates Spb4.

FIG. 8.

Disruption of DBP3 and DBP7, encoding two nonessential RNA helicases, is synthetically sick. (A) Yeast strains YPH499, Δdbp3, GAL-3xHA-Dbp7, Δdbp3/GAL-3xHA Dbp7, and GAL-3xHA Dbp6 were grown to early log phase, serially diluted (1, 10, 100, and 1,000 times) onto DEX (YPD) and GAL (YPG) plates, and incubated at 30°C. (B) Protein extracts made from yeast strains GAL-3xHA-Dbp3/Dbp7-TAP and Dbp7-TAP were immunoprecipitated (IP) using anti-HA antibodies conjugated to beads. Immunoprecipitated protein and total yeast extract (T; 5% of total protein extracted) were run on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were Western blotted with PAP antibodies (anti-TAP) and anti-HA antibodies (anti-HA).