Volume 14, Issue 3, Pages 331-342 (May 2004) The Carboxy-Terminal Extension of Yeast Ribosomal Protein S14 Is Necessary for Maturation of 43S Preribosomes  Jelena Jakovljevic, Pamela Antúnez de Mayolo, Tiffany D. Miles, Theresa Mai-Ly Nguyen, Isabelle Léger-Silvestre, Nicole Gas, John L. Woolford  Molecular Cell  Volume 14, Issue 3, Pages 331-342 (May 2004) DOI: 10.1016/S1097-2765(04)00215-1

Figure 1 Mutations in the Carboxy-Terminal Tail of rpS14 Affect the Growth Rate of Yeast Serial dilutions of yeast strains expressing wild-type RPS14B or the rps14b G131A, G132A, R134A, G135A, or R137A mutant genes were grown on C-ura-his solid medium containing galactose (left) or glucose (right) at 30°C for 2 days (top) or 6 days (bottom). In galactose medium, the wild-type RPS14A gene fused to the GAL promoter is also expressed. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)

Figure 2 Mutations in Residues in the Carboxy-Terminal Tail of rpS14 Cause 20S Pre-rRNA to Accumulate and Mature 18S rRNA to Be Diminished (A) Pathway of pre-rRNA processing in Saccharomyces cerevisiae. (B) rps14aΔ rps14bΔ pGAL1-RPS14A strains containing plasmid pRS313 bearing wild-type RPS14B or mutant rps14b were grown at 30°C in C-ura-his+gal medium to 1 × 107 cells/ml and shifted to C-ura-his+glu medium to shut off expression of wild-type rpS14 from pGAL1-RPS14A. Twelve hours after the medium transfer, RNA was extracted, subjected to gel electrophoresis, and stained with ethidium bromide. (C) RNA was extracted from strains described above in (B) 12 hr after the medium shift, and assayed by Northern analysis using oligonucleotide probes specific for each pre-rRNA processing intermediate or mature rRNA. U3 snoRNA was assayed as a loading control. Amounts of 18S rRNA in each mutant relative to the wild-type strain were quantified by phosphorimaging. (D) Wild-type RPS14B and rps14b R134A mutant yeast were grown at 30°C in C-ura-his+gal medium to 1 × 107 cells/ml and shifted to C-ura-his+glu for 5 hr. Cells were pulse-labeled for 2 min with [5,6-3H] uracil and chased with an excess of unlabeled uracil for the indicated times. RNA extracted at each time point was assayed by gel electrophoresis and autoradiography. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)

Figure 3 43S Preribosomes Containing 20S Pre-rRNA Accumulate in the rps14 Mutants (A) Whole-cell extracts were prepared from wild-type and rps14 mutant strains 12 hr after the carbon-source shift and subjected to centrifugation on sucrose gradients to separate preribosomes, 40S and 60S ribosomal subunits, 80S ribosomes, and polyribosomes. Fraction numbers are indicated for the gradient of wild-type extract. (B) Sucrose gradient profiles of 20S pre-rRNA, mature 18S rRNA, and the aberrant 17S pre-rRNA are shown from yeast expressing wild-type rpS14 (top panel) or the R134A mutant protein (bottom panel). RNA was extracted from 500 μl of each 1 ml fraction of the gradients shown in (A) and analyzed by Northern analysis using as a probe oligonucleotide 18S-A that hybridizes to both 20S pre-rRNA and 18S rRNA. (C) Extracts from yeast expressing wild-type rpS14 or the rps14 R134A mutant were prepared in buffer containing 4M KCl and subjected to centrifugation in 4 M KCl. (D) Sucrose gradient sedimentation profiles of ribosomal proteins rpS2 and rpL30 from yeast expressing the R134A mutant rpS14 (top panel), no rpS14 (middle panel), or wild-type rpS14 (bottom panel). Proteins in each fraction were detected by Western immunoblot analysis with a rabbit polyclonal antibody that binds to both rpS2 and rpL30. (E) Sedimentation profile of rpS14 from wild-type cells, R134A and R137A mutants, and the rps14 conditional null mutant. rpS14 in each fraction was detected by Western blotting using antibodies against rpS14. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)

Figure 4 Expression of the R134A Mutant rpS14 Results in Accumulation of 20S Pre-rRNA in the Cytoplasm The 20S pre-rRNA was detected by fluorescence in situ hybridization using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA were stained with DAPI (blue). RNA was assayed in strains containing GAL-RPS14 plus either the RPS14 wild-type gene or the R134A rps14 mutant gene. Cells were grown at 30°C in C-ura-his+gal medium and shifted to C-ura-his+glu medium for 12 hr. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)

Figure 5 The 17S rRNA Intermediate Present in the rps14 R134A Mutant Extends from the 5′ End of 18S rRNA to at Least 162 Nucleotides Upstream of the 3′ End of 18S rRNA RNA extracted from yeast expressing R134A mutant rpS14 was assayed by Northern analysis using a series of oligonucleotide probes complementary to sequences in the 18S rRNA. (A) All probes complementary to sequences downstream of nucleotide 1636 in 18S rRNA (162 nucleotides from the 3′ end of 18S rRNA) failed to hybridize to the 17S rRNA intermediate but did detect the 20S and 18S rRNAs. (B) All probes complementary to sequences upstream of nucleotide 1636 of 18S rRNA hybridized to 20S, 18S, and 17S rRNAs. (C) Cartoon depicting the relative positions of sequences complementary to each oligonucleotide probe. The structure is shown beginning with nucleotide 1158 in 18S rRNA (excluding nucleotides 1208–1451) and ending at the 3′ end of 18S rRNA. Helices 29, 30, 31, 42, 43, 44, and 45 of 18S rRNA are labeled. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)

Figure 6 The Carboxy-Terminal Extension of Ribosomal Protein S14 Is Located Near the 3′ End of 16S rRNA A model of the yeast 40S ribosomal subunit is shown, based on cryo-EM reconstruction and modeling of rRNA (yellow) and those yeast ribosomal proteins with bacterial or archaebacterial homologs of known 3D structure (green). Yeast rpS14 is shown in blue (the four carboxy-terminal amino acids are missing). Twenty of the last 23 nucleotides of 18S rRNA are shown, 17 in red, and the 3′-most nucleotides in white. The last three nucleotides of 18S rRNA could not be modeled from the bacterial structure. Molecular Cell 2004 14, 331-342DOI: (10.1016/S1097-2765(04)00215-1)