The putative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA processing through a direct interaction with Rps14

Abstract

One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3' end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.

FIG. 1.

Schematic representation of the pre-rRNA-processing pathway in the yeast Saccharomyces cerevisiae. RNA polymerase I transcribes the 35S pre-rRNA, which contains the mature rRNA sequences (18S, 5.8S, and 25S), ITS, and ETS. The 5.85 rRNA has both short (S) and long (L) forms. The 5S rRNA is transcribed by RNA polymerase III and joins 66S preribosomes in the nucleus. The positions of the oligonucleotides used in this study are indicated.

FIG.2.

Fap7 is required for processing of the 20S pre-rRNA at site D. (A) Growth rates of the UTP1, RPF1, and FAP7 depletion strains as well as that of the parental strain YPH499 in dextrose medium (YPD [Dex]). Growth was monitored for 24 h in YPD. (B to E) Northern analysis indicating that depletion of Fap7 blocks processing of the 20S pre-rRNA at site D but does not affect processing at A₀, A₁, or A₂. Exponentially growing depletion strains (GAL::3HA-UTP1, GAL::3HA-RPF1, and GAL::3HA-FAP7) and the parental strain (YPH499) were shifted from YPG/R to YPD, and cells were grown for 10 to 24 h. RNA was extracted from cells harvested at the indicated times, and equal amounts of RNA were resolved by 1.25% denaturing agarose gels (panels B to D) or 8% polyacrylamide-8 M urea gel (panel E), transferred to Hybond N⁺ membranes, and hybridized with several radiolabeled oligonucleotides (oligos) (panels B to D; indicated at the side of each panel). The positions of the various pre-rRNA species on the membranes are indicated on the left of each panel. Time points that were analyzed (h) and the strains used in the experiments are indicated on top of each panel. (F and G) Metabolic labeling indicating that depletion of Fap7 specifically inhibits processing at site D. The GAL::3HA-FAP7 and parental (YPH499) strains, both carrying a plasmid with a URA marker, were grown in galactose medium lacking uracil (SG/R-URA) to exponential phase and shifted to the nonpermissive dextrose medium (SD-URA), and after 5 h the cells were labeled with [5,6-³H]uracil for 2 minutes and then chased with an excess of cold uracil. Total RNA extracted from cells harvested at the indicated time points (in minutes) was analyzed by 1.2% denaturing agarose gels (panel F) and 8% polyacrylamide-8 M urea gel (panel G). The positions of the rRNA, pre-rRNA species, and tRNA are indicated on the left of each panel.

FIG. 3.

Fap7 is essential for 40S subunit synthesis but is not stably associated with 20S pre-rRNA and preribosomes. (A and B) Fap7 is required for the production of 40S ribosomal subunits. Extracts from GAL::3HA-FAP7 cells grown either in the permissive galactose medium only (panel A; YPG/R) or grown in the permissive medium and shifted to the nonpermissive dextrose medium for 5 h (panel B; YPD) were fractionated by 10% to 50% sucrose gradient centrifugation. The positions of ribosomal subunits, 80S ribosomes, and polysomes in the gradients were determined by measurements of absorption at 254 nm. (C) Sedimentation of 3HA-Fap7 in a 10% to 50% sucrose gradient. Extracts prepared from GAL::3HA-FAP7 cells grown in YPG/R were fractionated by 10% to 50% sucrose gradient centrifugation, and the sedimentation of 3HA-Fap7 in the gradient was determined by Western blot analysis. (D and E) Fap7-TAP does not coprecipitate the 20S pre-rRNA even when 20S pre-rRNA levels are increased by depleting Nob1. Strains (RIO2-TAP, GAL::3HA-NOB1, and FAP7-TAP) were grown in YPG/R to mid-log phase. The GAL::3HA-NOB1 and FAP7-TAP strains were subsequently shifted to YPD and grown for 6 h to deplete Nob1 (depleted). Rio2-TAP and Fap7-TAP were immunoprecipitated from extracts using immunoglobulin G-Sepharose beads. RNA extracted from 5 percent of the starting material and coprecipitated RNAs were resolved on 1.25% denaturing agarose gels. The 20S pre-rRNA was detected using oligonucleotide B. As negative controls, strains without TAP tags were used (lanes 2, 4 and 6 [−]). (E) Quantification of the results shown in panel D. The percentage of coprecipitated 20S pre-rRNA was determined using values generated from phosphorimager scans.

FIG. 4.

(A) Depletion of Fap7 causes accumulation of 20S pre-rRNA in the cytoplasm. FISH was performed on strains (YPH499 and GAL::3HA-FAP7) grown in galactose medium (YPG/R) (panels A to C and G to I) or dextrose medium (YPD) (panels D to F and J to L). TOPRO3 dye was used to stain nuclei (panels B, E, H, and K). FISH was performed using a Cy3-labeled D-A2 oligonucleotide that hybridizes to the 35S to 32S and 20S pre-rRNAs. (B) Fap7 depletion does not prevent Dim1-mediated dimethylation of the 20S pre-rRNA. Primer extension analysis on total RNA extracted from strains, indicated on top of the figure, grown in galactose-containing medium (YPG/R) only or grown in galactose-containing medium and shifted to dextrose-containing medium (YPD) for 5 hours. RNA was resolved on a 6% polyacrylamide-8 M urea gel and subjected to autoradiography.

FIG. 5.

Depletion of Fap7 results in accumulation of incompletely assembled 43S preribosomes. (A) Fap7 depletion alters the sedimentation behavior of 43S preribosome-associated proteins. Extracts prepared from exponentially growing GAL::3HA-FAP7 strains expressing TAP-tagged Rio2, Nob1, or Tsr1 (indicated on the right side of the panels) were fractionated by 10% to 50% sucrose density gradient centrifugation. Cells were either grown in YPG/R only (Undepleted) or grown in YPG/R and shifted to YPD for 5 h to deplete Fap7 (Depleted). The sedimentation of the TAP-tagged proteins was determined by Western blot analysis using the antibodies specific for the TAP tag. The positions of 40S and 60S subunits and of the 80S ribosomes/polysomes were determined by monitoring sedimentation of rRNAs in the gradient fractions by Northern analysis as shown in panel B. (B) The 20S pre-rRNA sediments at both 40S and 80S in Fap7-depleted cells. Northern blot analysis of RNA extracted from the sucrose gradient fractions is shown. The 25S rRNA was detected by hybridizing blots with radiolabeled oligonucleotide Y, whereas the 17S, 18S, and 20S rRNAs were detected using oligonucleotides B and A. (C) Fap7 depletion does not affect levels of Rio2, Tsr1, or Nob1 proteins. GAL::3HA-FAP7 strains expressing TAP-tagged Nob1, Rio2, or Tsr1 were grown in YPG/R and shifted to YPD for 5 h to deplete Fap7. The TAP-tagged protein and 20S pre-rRNA levels were monitored 0, 2.5, and 5 h after the shift to YPD (Dex). Rio2-TAP, Tsr1-TAP, and Nob1-TAP proteins (indicated at the top of each lane) were detected by Western blot analysis using an antibody (PAP) specific for the TAP tag. Mpp10 was used as a loading control. The 20S pre-rRNA was detected by Northern blot analysis using probe B.

FIG. 6.

Recombinant Fap7 directly interacts with wild-type and mutant (R134A) Rps14B proteins in GST pull-down assays. Six-histidine-tagged Fap7 and BSA were mixed with GST, GST-Rps14B, GST-Rps14B R134A, or GST-YOR287C and incubated on ice for 1 hour. Complexes were precipitated using glutathione-Sepharose beads and bound proteins (P) (lanes 2, 5, 8, and 11) were resolved by SDS-PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (I) (lanes 1, 4, 7, and 10) and 10% of the supernatants (S) (lanes 3, 6, 9, and 12) were also analyzed.

FIG. 7.

The putative ATPase motif of Fap7 is essential for cell viability. (A) Multiple sequence alignment of Fap7 with homologs from C. elegans (ceFap7; E02H1.6), human (TAF9/hFap7; ENSP00000261565), and A. gossypii (agFap7; ACL150W) Fap7. The alignment was generated using the DiAlign program (www.genomatix.de/cgi-bin/dialign/dialign.pl). The amino acid positions relevant to the substitution mutants used in panels B and C are marked with asterisks, and an explanation of the color coding is shown on the bottom of the figure. (B) Conserved amino acids in the Walker A and B motifs are essential for Fap7 function in vivo. Serial dilutions (10-fold) of GAL::3HA-FAP7 strains carrying p415GPD-FAP7 wild-type and mutant genes were grown in synthetic galactose medium (SG/R-LEU) and spotted on either dextrose-containing plates (SD-LEU [Dex]) or galactose-containing plates (SG/R-LEU [Gal/Raff]). Results from a plate incubated at 30°C are shown. (C) Conserved amino acids in the Fap7 Walker A and B motifs are essential for 20S pre-rRNA processing and 18S rRNA synthesis in vivo. GAL::3HA-FAP7 strains carrying p415GPD-FAP7 wild-type and mutant genes or the empty vector were grown in synthetic galactose medium to exponential phase and subsequently shifted to synthetic dextrose medium for 5 hours to deplete Fap7. RNA was extracted from cells harvested before (0 h) and after (5 h) the shift to dextrose medium and was analyzed by Northern blot analysis. The 20S pre-rRNA and 18S rRNA were detected using oligonucleotides B and A, respectively.