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	Figure 1. Demonstrated and proposed interactions between yeast U3 snoRNA and the pre-rRNA. (A) The proposed and experimentally demonstrated base pair interactions between yeast U3 snoRNA and the 18S part of yeast pre-rRNA. The three well demonstrated interactions (helices I–III) between box A and box A′ in U3 snoRNA and three segments of the 18S part of the pre-rRNA, which are involved in formation of the central pseudoknot structure in mature 18S rRNA (panel B), are shown (17,22,23,28). The putative helix IV which can be formed by U3 snoRNA and the fourth segment involved in the 18S pseudoknot structure (17) is also shown. The pre-rRNA sequences of interest are in green. The remaining parts of the molecule are schematically represented by green lines. The U3 snoRNA sequences of interest are in black, the remaining parts of the molecule are represented by black lines and schematically drawn stem–loop structures. Positions of nucleotides in the sequences are given as referred to the 5′ extremities of the molecules. The pre-rRNA segments involved in the 18S pseudoknot structure are in pink and yellow boxes, respectively, as in B. Positions of the A0, A1 and A2 cleavage sites in the pre-rRNA are indicated. (B) Schematic representation of the central pseudo-knot structure of yeast 18S rRNA (29,30) using the same colours as in A. (C) Schematic representation of the demonstrated and proposed interactions between yeast U3 snoRNA and the 5′ ETS region of yeast pre-rRNA. The well documented helix V (23,32,49), as well as the proposed helix VI (35), which correspond to the 5′- and 3′-hinge interactions in X. laevis, respectively (D), are shown, as well as the internal helix 1b′. (D) Schematic representation of the well documented 5′- and 3′-hinge interactions in X. laevis (35,36).
	Figure 2. Effects of mutations in the U3 snoRNA segment encompassing residues 49–72 on yeast cell growth. (A) The mutations generated in the yeast U3 snoRNA region extending from positions 49 to 72 and their effects on cell growth. Formations of helix V and the putative helix VI are represented at the top of the panel. The mutations generated in the S. cerevisiae U3A snoRNA are indicated below the WT sequence. Open triangle indicates a nucleotide deletion. The names of the mutations are given in the left column. The growth capabilities of cells expressing the variant U3 snoRNAs at 20, 30 and 37°C are indicated on the right side of the panel. +++, ++ and + correspond to normal, slightly decreased and strongly decreased growths, respectively, and − indicates a complete growth abolition. (B) Base substitutions in segment VI of U3 snoRNA strongly impair cell growth. The growth capability of JH84 cells transformed with the WT or a variant pASZ11::U3A plasmid (variants U3:VI-1, VI-2, VI-3, VI-4, VI-5 and VI-6) was tested at 20, 30 and 37°C on YPD medium, as described in ‘Materials and Methods’ section. A control experiment was performed with a JH84 cell transformed with an empty pASZ11 plasmid (−). Growth was examined after 48 h of incubation at 30 or 37°C and 72 h of incubation at 20°C. (C and D) Only the complete substitution of all residues in segment VI of U3 snoRNA has a strong effect on U3 snoRNA stability in cellulo. Analysis by northern blot (C) of the relative stabilities of the yU3A WT and variant RNAs in the JH84 S. cerevisiae cells grown at 30°C in YPD medium (see ‘Materials and Methods’ section) were performed using the 5′-end labelled oligonucleotide RT-yU3 as the probe and a 5′-end labelled oligonucleotide complementary to U6 snRNA (RT-yU6, see Supplementary Table S1) for normalization of the data. A graphic representation of the relative stabilities of variant U3 snoRNAs compared to WT U3 snoRNA is shown in D. The amounts of variant yU3A RNAs present in the cells were expressed as a percentage of the amount of WT RNA found in the control experiment (yU3mutant/yU6 relative to yU3WT/yU6). The values given are mean values from three independent experiments. Their standard deviations are represented by a bar.
	Figure 3. Cell growth is restored when mutations in the 5′-ETS region of pre-rRNA are compensated by mutations in segment VI of U3 snoRNA. (A) The WT helix VI is shown as proposed by Borovjagin and Gerbi (38), the variants of helix VI formed by compensatory substitutions in segment VI of U3 snoRNA and the 5′-ETS region are shown below, base substitutions are in grey. (B) Test for growth at 20 and 37°C on YPG medium. The NOY504 yeast strain was co-transformed with the WT or a variant pHW18 plasmid (WT, 5′-ETS:VI-1, 5′-ETS:VI-2, 5′-ETS:VI-5 and 5′-ETS:VI-6) containing one rDNA unit under the control of the GAL7 promoter and the WT or a variant pASZ11::U3A plasmid (WT, U3:VI-1, U3:VI-2, U3:VI-5 and U3:VI-6). The untransformed NOY504 strain (−) was used as a control. Growth capacities at 20 and 37°C (non-permissive temperature) were examine after 48 h of growth on YPG medium.
	Figure 4. Mutations in helix VI abolish pre-rRNA cleavages at sites A0, A1 and A2 and 18S rRNA production. (A) The organization of the yeast pre-rRNA encoded by the rDNA unit inserted in plasmid pHW18 (28,32,49) is shown in the upper part of the panel. The 5′- and 3′-ETS sequences and the ITS1 and ITS2 sequences correspond to thin lines, sequences corresponding to mature 18S, 5.8S and 25S rRNAs are shown by black rectangles, respectively. Positions of the A0, A1, A2 and A3 cleavages are indicated by arrows. The additional tag sequences in the 18S and 25S rRNAs are drawn as white boxes. Their complementary oligonucleotide probes 009 and 016 (44,45) are shown. The lower part of the Panel shows that mutations in fragment 5′-ETS VI of the yeast pre-rRNA abolish 18S rRNA production and production is restored by expression of a yeast U3 snoRNA containing compensatory mutations. NOY504 cells were co-transformed with the WT or a variant pHW18 plasmid (5′-ETS:WT, 5′-ETS:VI-1, 5′-ETS:VI-2 and 5′-ETS:VI-5), and the WT or a variant pASZ11::U3A plasmid (U3:WT, U3:VI-1, U3:VI-2 and U3:VI-5), as indicated above each lane. After 6 h of growth at non-permissive temperature (37°C) in YPG medium, total RNA was extracted, fractionated by electrophoresis on a 1.2% agarose–formaldehyde gel and transferred onto a nylon membrane. Oligonucleotides 009 and 016 (A and Supplementary Table S1) were used as the probes for northern blot analysis. Positions of the 18S and 25S rRNAs in the gel are indicated on the right side of the autoradiogram. The untransformed NOY504 strain (−) was used as a control. (B) The architecture of the yeast 35S pre-rRNA is schematically represented using the same symbols as in A. The pre-rRNA sequence complementary to the oligonucleotide probe 002 used for northern blot analyses and 008 used for primer extension are shown. The 23S and 20S pre-rRNA maturation intermediates and mature 18S rRNA are represented below together with the positions of cleavage sites and of the 002 and 008 target sequences. (C and D) Mutations in segment VI of yeast U3 snoRNA abolish cleavages at sites A0–A2. In C, northern blot analyses were performed on total RNAs extracted from JH84 cells transformed with the WT or a variant pASZ11::U3A plasmid (U3:VI-1, U3:VI-2 and U3:VI-5), after 24 h of growth at 30°C in YPD medium. In C total RNAs total RNAs were fractionated by electrophoresis on a 1.2% agarose–formaldehyde gel and transferred onto a Hybond+ membrane. Oligonucleotide 002 complementary to an ITS1 segment located upstream of site A2 (B) was used as the probe. The hybridization conditions are described in ‘Materials and Methods’ section. Total RNA extracted from JH84 cells transformed with an empty pASZ11 plasmid was used as a control, lane marked by (−). The results obtained reveal the absence of 20S production in JH84 cells expressing the variant U3 snoRNAs. In D, the same total RNAs as in B were analysed by primer extension using oligonucleotide 008 complementary to the 18S 5′-terminal segment as the primer. Conditions for primer extension are described in ‘Materials and Methods’ section. In parallel, total RNA extracted from JH84 cells transformed with the WT pASZ11::U3A plasmid was subjected to sequence analysis using oligonucleotide 008 as the primer. Products of the four sequencing reactions, Lanes U, G, C, A and the extension products were fractionated in parallel on a polyacrylamide sequencing gel. The extension products ending at site A0 are indicated by an arrow on the right side of the autoradiogram. The sequence read on the autoradiogram and the position of site A0 in this sequence are shown on the left side of the autoradiogram. The data obtained reveal the impairment of cleavage at site A0 when mutated U3 snoRNAs are expressed.
	Figure 5. Segment VI of U3 snoRNA is required for efficient association of proteins Mpp10, Imp4 and Imp3 in cellulo. (A) The JH84-SV10 cells (expressing the Flag-tagged Mpp10 protein and transformed with plasmid p413TEF::HA-Imp4p) and the JH84-SV3 cells (expressing the TAP-tagged Imp3 protein) were transformed with the WT or a variant pASZ11::U3A plasmid (U3:WT, U3:VI-1, U3:VI-2 and U3:VI-5). Cells were grown in YPG medium until stationary phase and then transferred in YPD medium for 24 h. They were washed with ice-cold water and lysed as previously described (20). A fraction of the extract (10%) was used to quantify the cellular amount of variant yU3A RNAs by northern blot analysis, using the same conditions as in Figure 2C (lanes 1–5). Another fraction of the extract (90%) was used for immunoselection assays, by incubation with beads coated either with an anti-Flag, an anti-HA antibody or with IgG as described in ‘Materials and Methods’ section. In both cases, the RNAs bound to the beads were extracted by proteinase K digestion followed by phenol extraction. The amount of the yU3A variant RNAs associated with the Flag-tagged Mpp10, HA-tagged Imp4 or TAP-tagged Imp3 protein was analysed by northern blot analysis as described above (Lanes 6–10). (B) The relative amounts of immunoselected variant yU3A RNAs, as compared to WT yU3A RNA were determined (IV/IWT%). As described in ‘Materials and Methods’ section, the binding capacity of the Flag-tagged Mpp10, HA-tagged Imp4 and TAP-tagged Imp3 proteins to the variant RNAs were expressed as a percentage of their binding capacity to the WT RNA (Flag-tagged Mpp10p, HA-tagged Imp4p and TAP-tagged Imp3p relative affinities indicated as percentages). The values given are mean values of three independent experiments and their standard deviations are represented by bars.

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