Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis

Abstract

The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

FIG. 1.

(A) Pre-rRNA processing in Saccharomyces cerevisiae. RNA polymerase I transcribes a 35S pre-rRNA that contains external (ETS) and internal (ITS) transcribed spacers. The pre-rRNA is subjected to chemical modifications and cleaved at several sites to produce the mature 18S rRNA, 5.8S rRNA, and 25S rRNA. Pre-rRNA processing can start with cleavage at site A₀, yielding the 33S pre-rRNA, followed by cleavages at sites A₁ and A₂ or A₃. Alternatively, cleavage at site A₃ occurs first, leading to the formation of the 23S and 27SA₃ pre-rRNAs, which are subsequently processed at sites A₀ (22S), A₁ (21S), and A₂ (20S), yielding the 20S pre-rRNA (43S preribosome). The 43S preribosome is exported to the cytoplasm, where it is cleaved at site D to generate the mature 18S rRNA and the SSU. Within the 66S preribosomes, the 27SA₂ is subjected to processing steps that lead to the production of 5.8S and 25S rRNAs. The 27SA₂ can be cleaved at site A₃, leading to the production of the 25S rRNA and the short form of 5.8S [5.8S_(S)]. Alternatively, the 27SA₂ pre-rRNA is processed at another site, yielding the long form of 5.8S [5.8S_(L)] and the 25S rRNA. These rRNAs join with 5S rRNA to form the mature 60S LSU. The oligonucleotides used in this study are indicated. (B) Schematic representation of conserved motifs in DEXD/H box helicases and explanation of their proposed function. The mutations that were introduced are indicated by arrows.

FIG. 2.

SSU RNA helicases associate with the SSU processome. The parental strain (YPH499) and strains expressing 3HA-tagged SSU RNA helicase proteins (Dhr1, Dhr2, Dbp8, Rrp3, Fal1, and Rok1) or 3HA-tagged LSU protein Rpf2 were grown in YP medium to exponential phase. Tagged proteins were immunoprecipitated from extracts using mouse anti-HA monoclonal antibodies (12CA5). Coimmunoprecipitated proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and coimmunoprecipitated RNA was resolved on 6% polyacrylamide-8 M urea gels. Mpp10 was detected with a rabbit anti-Mpp10 polyclonal antibody (9), whereas the U3 snoRNA was detected by Northern blotting with a radiolabeled antisense U3 oligonucleotide (9).

FIG. 3.

Overexpression of SSU RNA helicases from pYES2 plasmids. (A) SSU RNA helicases expressed from pYES2 plasmids accumulate at levels 5- to 50-fold higher than those of genomically encoded SSU RNA helicases. Yeast expressing genomically encoded TAP-tagged SSU RNA helicases under the control of the endogenous promoter (Genomic) or from pYES2 plasmids (pYES2) were grown in synthetic medium containing galactose and raffinose (SG/R-URA) to exponential phase. Extracts prepared from an equal amount of cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TAP-tagged SSU RNA helicases (indicated on top of each lane) were detected by Western blot analysis with an antibody (PAP) that recognizes the protein A portion of the TAP tag. Mpp10 was used as a control for loading of equal amounts of protein. (B) Mutations in conserved motifs in SSU RNA helicases do not dramatically affect protein expression and stability. Strains carrying SSU RNA helicase pYES2 plasmids were grown in synthetic medium containing dextrose to exponential phase and subsequently grown in synthetic medium containing galactose (SG/R-URA) for 6 h to induce protein expression. TAP-tagged wild-type or mutant RNA helicases (as indicated on top of each panel) were detected by Western blot analysis using an antibody (PAP) that recognizes the protein A domain in the TAP tag. In each lane, extracts prepared from an equal number of cells were loaded, and Mpp10 was used as a control for loading equal amounts of protein.

FIG. 4.

Motifs I and II of SSU RNA helicase mutants are frequently dominant negative. YPH499 strains carrying pYES2 plasmids encoding TAP-tagged wild-type or mutant SSU RNA helicases (indicated on the left of each panel) were grown in synthetic medium containing dextrose (SD-URA) to exponential phase and subsequently grown in synthetic medium containing galactose (SG/R-URA) for 6 h to induce protein expression. Serial dilutions (10-fold) were spotted on either dextrose-containing plates (SD-URA; DEX) to verify even spotting of the culture and on galactose-containing plates (SG/R-URA; GAL) to induce expression from the pYES2 plasmids. Growth was monitored at 17°C, 23°C, and 30°C.

FIG. 5.

Motif I and II SSU RNA helicase mutations are generally lethal, whereas the Q motif and motif III mutations are frequently tolerated. SSU RNA helicases controlled by the tetO7 promoter are efficiently depleted when doxycycline is added to medium. Cells were grown in dextrose-containing media (YPD) to exponential phase, and serial dilutions (10-fold) of tetO7 strains were spotted on either glucose-containing plates (−DOX) (left panels) or glucose containing plates with doxycycline (+DOX) (right panels) and incubated at 30°C. Serial dilutions (10-fold) of SSU RNA helicase tetO7 conditional strains harboring pYES2 plasmids encoding TAP-tagged wild-type and mutant RNA helicases (indicated on the left of each panel) were grown in synthetic dextrose medium (SD-URA) and spotted on glucose-containing plates (SD-URA; DEX) and galactose-containing plates with doxycycline (SG/R-URA plus DOX; GAL + DOX). Growth was monitored at 17°C, 23°C, and 30°C.

FIG. 6.

Growth curves of strains expressing SSU RNA helicases from pYES2 plasmids. Growth rates of the wild type (diamonds), motif I K-A mutants (squares), motif I K-R mutants (triangles), and motif II D-A mutants (stars) in SG/R-URA at 23°C are shown. Strains carrying pYES2 RNA helicase plasmids (indicated in the legends on the figure) were grown in synthetic dextrose-containing medium (SD-URA) to exponential phase and subsequently shifted to galactose-based medium (SG/R-URA); growth was monitored for 24 to 48 h at 23°C. The absorbance at an optical density of 600 nm is plotted on the y axis, whereas the time in galactose-based medium (in hours) is plotted on the x axis.

FIG. 7.

Overexpression of dominant negative SSU RNA helicases causes aberrant pre-rRNA processing. Strains carrying pYES2 RNA helicase plasmids (indicated on the top of each panel) were grown in synthetic dextrose-containing medium (SD-URA) to exponential phase and subsequently shifted to galactose-based medium (SG/R-URA). Yeasts were grown for 24 to 48 h at 23°C. RNA was extracted from cells harvested at the indicated time points (in hours), and equal amounts of RNA was resolved by 1.25% denaturing agarose gels. Northern blots were hybridized with various radiolabeled oligonucleotides (indicated on the sides of the panels). The positions of the various pre-rRNAs on the membrane are depicted on the left of each panel.