Ribosome Structure and Activity Are Altered in Cells Lacking snoRNPs that Form Pseudouridines in the Peptidyl Transferase Center  Thomas H. King, Ben Liu, Ryan R. McCully, Maurille J. Fournier  Molecular Cell  Volume 11, Issue 2, Pages 425-435 (February 2003) DOI: 10.1016/S1097-2765(03)00040-6

Figure 1 The PTC Contains Six Ψs, Targeted by Five H/ACA Guide snoRNAs (A) Secondary structure of the central loop region of the yeast PTC (domain V) showing the six Ψs of interest. The Ψs are identified with different colors, and Nms are green. Ψ2822, orange; Ψ2861, purple; Ψ2876, blue; Ψ2919, red; Ψ2940, yellow; Ψ2971, cyan. (B) The predicted 3D arrangement of modified nucleotides in the PTC. The yeast Ψ and Nm modifications have been painted onto the structure of the Haloarcula marismortui PTC, complexed with CCdA-p-puromycin (Yarus analog, magenta) (Nissen et al., 2000). The color scheme is the same as in (A). (C) Northern blot analysis of snoRNA levels in wt cells (+) or cells with five guide snoRNA genes disrupted (−). (D) Ψ modification status of wt cells (WT) or cells lacking five guide snoRNAs (−6Ψ). Total RNA was treated with CMC and analyzed by a primer extension reaction. Extension of cDNA stops 1 nt before the Ψ. (+), CMC treated; (−) untreated. Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 2 Cells Lacking Ψs in the PTC Are Growth Impaired (A) Growth properties of test cells on solid medium. Growth after 2 days on YPD medium is shown for cells lacking 6Ψs or only Ψ2919; wt, wild-type cells. Images of isolated single colonies are also shown. (B) Growth of cells lacking 6Ψs or only Ψ2919 in liquid medium at 30°C. Data are also shown for a wild-type control strain that is the progenitor of all test strains except (−) Ψ2919 (YS602), and the isogenic parent of strain (−) Ψ2919 (YS625). (C) Competitive growth analysis was carried out by mixing equal numbers of (−) 6Ψ and wt cells in YPD at 30°C. Results are shown for five complete cycles of growth. The proportion of mutant cells was determined by plating on selective and nonselective medium. (D and E). Sensitivity to paromomycin is increased in cells lacking Ψ in the PTC. (D) Solid medium. Midlog cultures of (−) 6Ψ, (−) Ψ2919, and wt cells were diluted serially (1:5), spotted onto YPD containing 0.3 mg/ml paromomycin or YPD lacking paromomycin, and incubated for 2 days at 30°C. (E) Liquid medium. Cultures started from fresh YPD inocula were grown in YPD containing 0.4 mg/ml paromomycin at 30°C. Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 3 Cells Lacking Ψs in the PTC Are Translationally Impaired (A) In vivo production of luciferase. Cells lacking 1, 5, or 6Ψs were transformed with a plasmid-encoded GAL::luciferase gene, and grown in galactose medium. Light production from luciferase was measured in a luminometer. Samples contained approximately equal numbers of cells. (−) 6Ψ, all Ψs depleted; (−) 5Ψ, all Ψs depleted except Ψ2919; (−) Ψ2919 depleted; and WT, wild-type cells. (B) In vivo incorporation of [35S]methionine in (−) 6Ψ cells, and in (−) 5Ψ cells. Error bars correspond to standard deviation values for triplicate measurements. (C) Translational impairment in cells lacking Ψs in the PTC. Rates of [35S]methionine incorporation in vivo are compared with wild-type control cells. Cells dependent on snR10ΔC process rRNA normally but are unable to make Ψ2919 (Figures 5 and 6). (D) Polysome patterns are altered in cells lacking Ψs. Early log phase cells were chilled in the presence of cycloheximide, lysed, and extracts were analyzed on 7%–50% sucrose gradients. Absorbance profiles (A254 nm) are shown for extracts from wt cells, (−) 6Ψ cells, and (−) Ψ2919 cells. Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 4 Ribosomes Lacking Ψs in the PTC Are Structurally Altered (A) Cells lacking all six Ψs were treated with dimethylsulfate (DMS) for 2 min. PTC bases modified by DMS were identified by a primer extension assay. Right panel, cDNA patterns for a region exhibiting differences in reactivity to DMS. (−) 6Ψ, ribosomes from cells lacking all six Ψ modifications; wt, wild-type ribosomes. The concentration of DMS (mM) is indicated above each lane. Left panel, DNA sequence ladder of the affected region (generated from a rDNA template). (B) Secondary structure of the yeast PTC and the sarcin/ricin domain of LSU rRNA, with DMS-reactive nucleotides shown. Asterisks, bases reactive with DMS in both wt and mutant cells. Closed arrowheads, nucleotides with increased DMS reactivity in mutant cells. Predicted H bonds between nucleotides in domains V and VI are indicated (solid lines) (Ban et al., 2000). Uridines converted to Ψs in wild-type cells are boxed. Roman numerals, standard rRNA domains. SRL, sarcin/ricin loop. (C) DMS modification pattern for nucleotides in domain VI. Reaction of A3003 was detected by two different primers (bottom panels). Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 5 A Point Mutation Separates snR10 Modification and Processing Functions (A) Location of a C residue deleted from snR10 (↓). (B) A 1 nucleotide deletion in snR10 abolishes formation of Ψ2919. Primer extension results used to detect formation of Ψ2919 and a control modification (Ψ2940). Northern analysis showed that snR10 accumulation was not affected by the point mutation (bottom bands). (C) Cells lacking Ψ2919 grew as well as wild-type cells in the absence or presence of paromomycin (paro). A series of 10-fold diluted cells were spotted on SC-Ade plates and incubated for two days at 30°C. WT, wild-type cells; (−) Ψ2919, cells without snR10; (−) 6Ψ, cells without all five guide snoRNAs; + WT snR10, (−) Ψ2919 cells with wild-type snR10 expressed ectopically; + snR10ΔC, (−) Ψ2919 cells with snR10ΔC. Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 6 Loss of Ψ2919 Impairs the Function but Not Production of Ribosomes (A) Relative amount of ribosomal subunits are shown for wild-type cells (WT), cells lacking snR10 (− snR10), and cells with the snR10ΔC variant (snR10ΔC). The ratio of 60S to 40S subunits is shown for each strain (top). (B) Half-mer polysomes form in cells harboring the snR10ΔC mutation. (C) Inactivation of Ψ2919 formation impairs translation activity. Cells lacking snR10 or producing only snR10ΔC have lower rates of [35S]methionine incorporation in vivo (see also Figure 3C). Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)

Figure 7 Two Ψs in the PTC Are in Close Proximity to Binding Sites of A and P Site tRNAs (A) Ψ2919 occurs in the A loop. The rRNA bases that interact with the 3′ end of tRNA bound to the A site are shown, using the structure and numbers of the H. marismortui ribosome. Nucleotide numbers for S. cerevisiae are in parentheses. U2589 (red) corresponds to yeast Ψ2919. The A site tRNA analog is shown in magenta. (B) Ψ2861 is in close proximity to nucleotides in the P loop. The nonbridging phosphate oxygen of Ψ2861 is 3.6 Å from G2283, which is believed to form a H bond with the 3′ terminal A of P site tRNA (Nissen et al., 2000). Molecular Cell 2003 11, 425-435DOI: (10.1016/S1097-2765(03)00040-6)