S. cerevisiae U2 snRNA. A partial sequence and two alternate secondary structures of yeast U2 are shown. The three constitutive pseudouridines (Ψ35, Ψ42 and Ψ44) as well as the two inducible pseudouridines (Ψ56 and Ψ93) are also shown. The Sm-binding site (shaded boxes) and the branch site recognition sequence (underlined) are indicated. According to Hilliker et al (2007) and Perriman and Ares (2007), toggling between two alternate structures, stem-loop IIa and stem IIc, is required for splicing.

Conditionally inducible formation of Ψ56 and Ψ93. (A) S. cerevisiae cells were grown in YPD to various ODs (indicated at the top), total RNA was recovered and pseudouridylation was assayed (by CMC modification followed by primer extension). Samples of even-numbered lanes were treated with CMC and samples of odd-numbered lanes were not treated with CMC. Signals corresponding to Ψ35, Ψ42, Ψ44, Ψ56 and Ψ93 are indicated. U2 sequencing is also shown on the left. (B) S. cerevisiae cells were first grown at 30°C, then were kept at 30°C (lanes 1 and 2) or shifted to 37°C (lanes 3 and 4) or 45°C (lanes 5 and 6) for 30 min. Total RNA was recovered and pseudouridylation assay (CMC modification followed by primer extension) was performed (see (A)). (C) RNA was recovered from cells at various OD₆₀₀ (indicated). U2 snRNA was site-specifically radiolabelled at position 93 (see Materials and methods). After digestion with nuclease T2, cleaved RNA samples were analysed by thin layer chromatography (TLC). Relative percentages of pseudouridylation at position 93 were calculated (under each panel).

Pus7p and snR81 RNP are required for the formation of Ψ56 and Ψ93, respectively. (A) The sequence and secondary structure of snR81 box H/ACA RNA are shown. Boxes H and ACA are indicated (shaded boxes). The two internal loops, including the 5′ one that guides the formation of Ψ42 in U2 and the 3′ one that directs the formation of Ψ1051 in 25S rRNA, are also shown. (B) Sequences surrounding Ψ35 (target of Pus7p) and Ψ56 of U2 are aligned and sequences flanking Ψ1051 of 25S rRNA (target of 3′ pocket of snR81) and Ψ93 of U2 are also aligned. Identical (a vertical line) and different (X) nucleotides are indicated. (C) Wild-type (BY4741) (lanes 1 and 2), snr81Δ (lanes 3 and 4), pus1Δ (lanes 5 and 6), pus7Δ (lanes 7 and 8) and cbf5D95A (lanes 9 and 10) strains were all grown to saturation. CMC-primer-extension-based pseudouridylation assay was then carried out (see legend to Figure 2A). Signals corresponding to Ψ35, Ψ42, Ψ44, Ψ56, Ψ93 and Ψ94 are indicated.

Induced pseudouridylation at positions 56 and 93 is independent of prior constitutive pseudouridylation at other sites. (A) snR81 RNA, containing a mutated 3′ pseudouridylation pocket (lanes 1 and 2) or a mutated 5′ pseudouridylation pocket (lanes 3 and 4), was expressed in an snr81-deleted strain. Upon entry into stationary phase, cells were collected, RNA isolated and pseudouridylation assay (CMC modification followed by primer extension) was carried out. In lanes 1 and 3, CMC was omitted; in lanes 2 and 4, samples were treated with CMC. Signals corresponding to Ψ35, Ψ42, Ψ44 and Ψ93 are indicated. (B) Pseudouridylation assay as in (A). Instead of expressing a snR81 mutant, wild-type U2 (lanes 1, 2, 5, 6, 9, 10, 13 and 14) and two mutant U2 substrates were expressed in the cell. In lanes 3, 4, 7 and 8, U2 contained a U33A mutation; in lanes 11, 12, 15 and 16, U2 contained a U40G mutation. Before RNA isolation, cells were allowed to grow to log phase (lanes 1–12, OD2) or stationary phase (lanes 13–16, OD15). In lanes 5–8, cells were further heat shocked before being lysed for RNA isolation. Samples of even-numbered lanes were treated with CMC and samples of odd-numbered lanes were not treated with CMC. Signals corresponding to Ψ35, Ψ42, Ψ44, Ψ56 and Ψ93 are indicated.

Imperfect interactions between enzyme and substrate are necessary for induced pseudouridylation. (A) Shown are interactions between the wild-type 3′ pocket of snR81 and the wild-type and various mutant U2 substrates. (a) Wild-type U2; (b) U2 with U98A/U102A mutations; (c) U2 with U94A/U98A/U102A mutations; (d) U2 with an A96U mutation; (e) U2 with A96U/U98A mutations; (f) U2 with A95C/A96U mutations; (g) U2 with A95C/A96U/U98A/U102A mutations. Bold and italicized letters represent mutations. (B) Pseudouridylation assay (CMC modification followed by primer extension) upon expression of wild-type U2 (a) (lanes 1, 2, 7 and 8), U2 with U98A/U102A mutations (b) (lanes 3, 4, 9 and 10) and U2 with U94A/U98A/U102A mutations (c) (lanes 5, 6, 11 and 12). Lanes 1–6, total RNA was isolated from log-phase cells; lanes 7–12, total RNA was isolated from stationary-phase cells. (C) Pseudouridylation assay as in (B) except that different mutant U2 RNAs (as indicated at the top of each lane) were used. In lanes 1–10, log-phase cells were used; in lanes 11–20, stationary-phase cells were used. (D) Interactions of mutant 3′ pseudouridylation pocket of snR81 with various U2 are shown. (h) snR81 with U101A/U105A mutations interacting with wild-type U2; (i) snR81 with U101A/U107A mutations interacting with wild-type U2; (j) snR81 with U101A/U105A mutations interacting with U2 containing U98A/U102A mutations; (k) snR81 with U101A/U107A mutations interacting with U2 containing A96U/U98A mutations. Mutations are indicated by bold and italicized letters. (E) Pseudouridylation assay as in (B) except that various snR81 RNAs and U2 RNAs, as indicated at the top of each lane, were used. In lanes 1, 2, 5, 6, 9, 10, 13 and 14, log-phase cells were used; in lanes 3, 4, 7, 8, 11, 12, 15 and 16, stationary-phase cells were used.

Ψ93 formation contributes to splicing regulation. (A) Yeast cup1Δ strain was transformed with a plasmid carrying an ACT1–CUP1 reporter pre-mRNA gene (either wild-type, lanes 1 and 2, or gAG mutant, lanes 3–5) (Hilliker et al, 2007), along with either a vector only (lanes 1 and 3) or a plasmid containing snR81 (U101A/U105A) gene specifically targeting position 93 of U2 (lanes 2 and 4). Cells (for lanes 1–4) were harvested during early/mid-log phase. Total RNA was recovered and primer extension was carried out (using an ACT1–CUP1-specific primer and a U6-specific primer), allowing for checking pre-mRNA splicing efficiency. In lane 5, cells were transformed with only the gAG/A mutant pre-mRNA (no guide RNA co-transformation), but the cells were harvested in stationary phase (OD₆₀₀=20) (rather than in log phase). Primer-extension products corresponding to pre-mRNA, mRNA, lariat and U6 are indicated. (B) Splicing efficiency was quantified, based on three independent experiments. mRNA level relative to U6 level (upper panel), and the ratio of mRNA to (mRNA+lariat), reflecting the efficiency of second step of splicing (lower panel), are shown. (C) cup1Δ cells, transformed with the gAG/A mutant pre-mRNA along with an empty vector or a plasmid containing an artificial box H/ACA guide RNA gene specifically targeting position 93 of U2 (corresponding to lanes 3 and 4 in (A, B)), were plated on a synthetic medium containing no copper ([Cu²⁺]=0) or 0.08 mM copper ([Cu²⁺]=0.08).