Strong dependence between functional domains in a dual-function snoRNA infers coupling of rRNA processing and modification events

Abstract

Most small nucleolar RNAs (snoRNAs) guide rRNA nucleotide modifications, some participate in pre-rRNA cleavages, and a few have both functions. These activities involve direct base-pairing of the snoRNA with pre-rRNA using different domains. It is not known if the modification and processing functions occur independently or in a coordinated manner. We address this question by mutational analysis of a yeast box H/ACA snoRNA that mediates both processing and modification. This snoRNA (snR10) contains canonical 5'- and 3'-hairpin structures with a guide domain for pseudouridylation in the 3' hairpin. Our functional mapping results show that: (i) processing requires the 5' hairpin exclusively, in particular a 7-nt element; (ii) loss of the 3' hairpin or pseudouridine does not affect rRNA processing; (iii) a single nucleotide insertion in the guide domain shifts modification to an adjacent uridine in rRNA, and severely impairs both processing and cell growth; and (iv) the deleterious effects of the insertion mutation depend on the presence of the processing element in the 5' hairpin, but not modification of the novel site. Together, the results suggest that the snoRNA hairpins function in a coordinated manner and that their interactions with pre-rRNA could be coupled.

Figure 1.

Yeast pre-rRNA processing pathway. The major rRNA precursors and final products are shown. Cleavage sites relevant to the present study are indicated, as well as the approximate location of the Ψ guided by snR10 in 25S rRNA.

Figure 2.

A 7-nt sequence element in snR10 is required for normal cell growth and pre-rRNA processing. (A) Schematic structure of the snR10 snoRNA. Key features are identified, including the H and ACA boxes, the Ψ guide region and five segments complementary to pre-rRNA. The complementary segments are numbered and highlighted with lines outside the snoRNA structure. Segment 2, which is complementary to the 5′ETS (thick line), is revealed in the present study to be essential for the processing function of snR10. Sites within and neighboring this segment were subjected to mutational analysis; the flanking sites are identified with dashed lines (Supplementary Table S1). A lower panel shows complementarity between segment 2 and the 5′ETS of pre-rRNA. The 7-nt element required for processing is boxed. (B) Mutation of segment 2 impairs cell growth. Test cells expressing snR10 with mutations were serially diluted (1:5), dotted on solid medium lacking Trp, and incubated at 30°C for 30 h. Control cells include: a wild-type strain (YS602) with an empty vector (Con.), and a parental test strain depleted of endogenous snR10 that contains either an empty vector (−snR10) or a plasmid that expresses wild-type snR10 (+WT). Growth properties of cells expressing snR10 variants with mutations in segment 2 or 4, (M2 or M4), are shown. The arrow above the colony patterns indicates decreasing numbers of cells. (C) Mutation of segment 2 causes accumulation of a 23S precursor to 18S rRNA: this product is defined by the 5′-end of the transcript and the A₃ cleavage. Total RNA was analyzed by northern hybridization. U2 snRNA is shown as a loading control. (D) Polysome patterns are altered by mutation of segment 2. Whole-cell extracts containing similar amounts of RNA were separated by sucrose gradient fractionation and ribosomal complexes detected by UV absorbance. The identities of ribosomal complexes are indicated for control cells (Con.). Reductions in the level of free 40S subunits are marked by open arrowheads and increases in the level of free 60S subunits by asterisks.

Figure 3.

The 7-nt processing element in snR10 is single-stranded. (A) Secondary structure of the 5′-hairpin region in snR10 that contains the 7-nt processing element. The structure was predicted with the folding program Mfold, confirmed and refined using the in vivo chemical probing data in (B) below. The relative extent of DMS modification is indicated by the number of asterisks. The seven nucleotides required for pre-rRNA processing are circled. The potential H box is indicated. (B) The 7-nt element is present in an internal loop of the 5′ hairpin. Following DMS modification in vivo, the structure of snR10 was probed by primer extension analysis with extension products separated on an 8% sequencing gel, next to sequencing products created with the same primer. Nucleotides accessible to DMS are identified.

Figure 4.

The processing function of snR10 is mediated by its 5′ hairpin. (A) The roles of the 5′ and 3′ hairpins in snR10 function were examined with hybrid snoRNAs containing hairpins from snR10 and snR36 in test cells lacking snR10. The structures of the hybrid snoRNAs are depicted schematically, with the snR10 portion shown with thick lines and the snR36 portion with thin lines. (B) The hybrid snoRNAs are expressed at normal levels as evident in northern hybridization results. (C) Normal growth occurred with the hybrid containing the 5′ hairpin of snR10 (H10/36), but not the hybrid containing the 3′ hairpin of snR10 (H36/10). The snoRNA snR38 was used as a control for loading. (D) A normal ribosome–polysome pattern was restored by the hybrid snoRNA with the 5′ hairpin from snR10 (H10/36). Half-mer polysomes are evident in test cells that express a snR10 mutant lacking a C nucleotide (M-C) in the guide domain; this deletion blocks Ψ2923 formation (12). Accumulation of half-mer polysomes is indicated by an arrow. (E) The Ψ2923 modification is formed with a hybrid snoRNA containing the 3′ hairpin of snR10 (H36/10). Total RNA was treated with CMC and Ψ detected by primer extension analysis. The extension stops representing Ψ2923 and Ψ2880 (guided by snR10 and snR34, respectively) are identified.

Figure 5.

A 1-nt insertion mutation in the Ψ guide domain severely impairs cell growth. (A) Sequences of three mutant variants of snR10 snoRNA. The uridine normally targeted in 25S rRNA (U2923) is circled and an adjacent uridine that is modified with the two insertion variants of snR10 is marked with an arrow (U2924). In one mutant, a single C (italicized) has been deleted (M−C). Two other mutants contain C and CA insertions at the same position, as indicated by an arrow (M + C, M + CA). (B) Northern hybridization results show that the two insertion mutant snoRNAs are expressed at comparable levels. (C) The shift in Ψ modification site for the insertion mutations is shown by primer extension data, as in Figure 4, and the positions of the natural and novel Ψs are indicated. (D) The C-insertion mutation severely impairs cell growth (M + C). Growth was examined in a serial dilution assay as in Figure 2B.

Figure 6.

The single nucleotide insertion in the guide domain causes a strong decrease in rRNA levels. (A) Northern hybridization probing of rRNA species reveals sharp reductions of ∼60–80% in the level of mature rRNAs, and significant accumulation of 35S and 20S pre-rRNAs. (B) The steady-state levels of 18S, 5.8S and 25S rRNAs are dramatically reduced as indicated by measuring the hybridization signals in (A), and normalizing to the amount of 5S rRNA. (C) The 20S pre-rRNA with Ψ at the novel site accumulates normally in the cytoplasm, as indicated by the presence of dimethylation modifications, which are formed only in cytoplasmic 20S pre-rRNA. The methylation modifications were detected by primer extension analysis.

Figure 7.

rRNA processing is severely impaired by the single nucleotide insertion in the Ψ guide domain. (A) rRNA processing was examined by in vivo pulse-chase labeling. Test cells were labeled with [methyl-³H]methionine, and total RNA was prepared during a chase with unlabeled methionine. RNA was separated in a 1.2% agarose gel, transferred to a membrane, and the radioactive bands visualized with a phosphorimager. Normal rRNA species are identified by arrows and unusual pre-rRNAs are marked with arrowheads. (B) The C-insertion mutation causes an abnormal polysome pattern (M + C). Polysome profiles were analyzed as in Figure 2. The abnormal 50S complex in the mutant cells is indicated by an arrowhead.

Figure 8.

A change in rRNA structure accompanies the shift in Ψ modification. (A) Secondary structure of the A-loop region of the PTC region in 25S rRNA. The arrow identifies a nucleotide (C2889) with reduced DMS reactivity, as shown in (B). The normally modified Ψ2923 site and new site of modification (U2294) are circled. (B) Primer extension probing of rRNA structure in the A-loop region. Total RNA was prepared from cells treated with DMS and subjected to primer extension mapping. A nucleotide (C2889) showing altered DMS accessibility in test cells is marked with an arrow. A control nucleotide (A2847) sensitive to DMS treatment in all test strains is marked with an arrowhead.

Figure 9.

The severe growth defect caused by the C-insertion mutation is not due to mis-targeting of the Ψ modification. (A) Construction of a hybrid snoRNA with the mis-targeting guide domain from snR10 (H36/10 + C). The mutant snoRNA includes the 5′ hairpin of snR36 (thin line) and 3′ hairpin of snR10 (thick line) with the C-insertion mutation. (B) The levels of Ψ modification at the novel site are similar for both the novel hybrid and the initial snR10 variant with the C-insertion mutation (H36/10 + C and M + C), as shown by primer extension data. (C) The hybrid snoRNA with the C-insertion in the guide domain causes moderate growth defects similar to those seen for snR10 depletion. (D) Similar moderate defects in processing are also apparent for the hybrid snoRNA with the C-insertion mutation and for the snR10-depletion condition. (E) Structure of a double mutant of snR10 that includes changes in both the processing and modification hairpin domains (M2 + C). The changes are: substitution mutations in segment 2 of the 5′ hairpin, and the C-insertion in the guide region in the 3′ hairpin. (F) A 4-nt substitution in the 7-nt processing element suppresses the growth defect caused by the 3′ C-insertion mutation. (G) Co-expression of hybrid snoRNAs H10/36 and H36/10 + C in snR10-depleted cells. Total RNA was analyzed by northern hybridization using probes specific to the 5′ half (upper panel), or the 3′ half (middle panel). 5S rRNA was used as a control for loading. (H) No growth defect resulted from co-expression of the H10/36 and H36/10 + C snoRNAs.