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FIGURE 1. Post-transcriptional modification at the branch point recognition region of U2 snRNA from different species. The recognition region is highlighted with a dashed line. Positions examined in this and previous studies are shadowed. Proposed interactions between the tested positions are indicated.
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FIGURE 2. Pseudouridylation of yeast U2 snRNA induced by human SCARNA15. (A) Predicted base-pairing of huSCARNA15 with human and yeast U2 snRNA. Point mutations introduced into huSCARNA15 are shown in red. (B) Mapping of pseudouridines by fluorescent primer extension reactions. Top (gray) trace: Wild-type S. cerevisiae (BY4741) U2 snRNA is normally modified at positions 44, 42, and 35. Traces 2 (red) and 3 (green): When huSCARNA15 was expressed in the wild-type (red) or mutant pus7Δ strains (green), position 40 became pseudouridylated, as indicated by the star. Trace 4 (light blue): In the snr81Δ strain, huSCARNA15 induces pseudouridylation of positions 40 and 38 (stars). Traces 5 (magenta) and 6 (violet): 1-nt insertions between the box ACA and the upper stem (+U@ACA, or +A@loop) eliminate modification at position 38. Trace 7 (blue): Control snr81Δ strain without exogenous guide RNA. Traces 8–10: When expressed in the snr81Δpus1Δ mutant strain, the mutated RNAs produce only Ψ40 (star).
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FIGURE 3. Pseudouridylation of yeast U2 snRNA induced by Xenopus SCARNA15 and by the C. elegans ortholog, snoRNA C50C3.14. (A,C) Predicted base-pairing between SCARNA15 and Xenopus U2 snRNA (A) or C. elegans U2 snRNA (C). The equivalent yeast U2 snRNA sequences are shown below. Mutated nucleotides are highlighted in red. (B,D) Fluorescent primer extension reactions. (B) Traces 1 (green) and 2 (blue): Wild-type Xenopus SCARNA15 modifies yeast U2 snRNA at positions 42 and 40 when expressed in the snr81Δ mutant yeast strain. Trace 3 (red): Chimeric SCARNA15 (human 5′ terminal hairpin and Xenopus 3′ terminal hairpin) modifies only position 40 of yeast U2 snRNA. Trace 4 (violet): Mutation 1 alone did not change pseudouridylation of position 38. Traces 5 (light blue) and 6 (magenta): A combination of mutations 1 and 2 is required to make the Xenopus 3′ terminal pseudouridylation pocket functional at position 38 in U2 snRNA. Trace 7 (dark violet): Mutation 3 makes the 3′-terminal domain of Xenopus SCARNA15 nonfunctional on yeast U2 snRNA; only the 5′ terminal pocket functions to position Ψ42. Stars indicate induced modifications. (D) Traces 1 (red) and 2 (blue): Both wild-type (red) and mutant C. elegans SCARNA15 (blue) are fully functional at all three predicted positions in yeast U2 snRNA: Ψ42, Ψ40, and Ψ38 (stars).
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FIGURE 4. Pseudouridylation of yeast U2 snRNA induced by Xenopus and human SCARNA4. (A) Predicted base-pairing of SCARNA4 with vertebrate and yeast U2 snRNA. A point mutation in human SCARNA4 that converts the A–G mismatch into a canonical A–U base pair is shown in red. A mutated Xenopus SCARNA15 that mimics base-pairing of human SCARNA4 with U2 snRNA is also shown; mutated positions are highlighted in red. (B) Fluorescent primer extension reactions. Traces 1 (green), 2 (blue), and 3 (pale brown): Xenopus and human SCARNA4 rescue yeast U2 snRNA pseudouridylation at position 42 (star) in the snr81Δ and snr81Δpus1Δ mutant strains. Trace 4 (red): Human SCARNA4 shows no activity on position 40 unless G in the 3′-terminal pocket is mutated to U. Trace 5 (dark green): A–G base-pairing within the 3′ terminal pseudouridylation pocket of the mutated Xenopus SCARNA15 makes the 3′-terminal domain nonfunctional on yeast U2 snRNA. Note absence of Ψ40. Traces 6 (dark blue) and 7 (brown): Controls without exogenous RNAs.
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FIGURE 5. Pseudouridylation of U2 snRNA at position 44 interferes with pseudouridylation at position 45 in the yeast cell system. (A) Base-pairing of wild-type and mutated variants of Drosophila scaRNA:ΨU2-35.45 with yeast U2 snRNA. Mutated nucleotides are shown in red. (B–G) Fluorescent primer extension reactions. (B) Traces 1 (green) and 2 (light brown): Drosophila scaRNA:ΨU2-35.45 cannot induce pseudouridylation at position 45 when expressed stepwise from an inducible Gal promoter in the pus7Δ yeast strain. Rescue of pseudouridylation at position 35 (star) serves as an internal positive control. Traces 3 (blue) and 4 (dark blue): In the same experimental setup, Drosophila scaRNA:ΨU2-35.45 is active at position 45 (star) in the pus1Δ strain, which has no Ψ44 in U2 snRNA. (C) Trace 1 (magenta): scaRNA:ΨU2-35.45 optimized for positioning Ψ45 in yeast U2 snRNA efficiently modifies position 45 in wild-type yeast strain only when overexpressed. Trace 2 (green): The same yeast-optimized RNA modifies position 45 inefficiently in Pus1p-positive strains, such as pus7Δ. Trace 3 (blue): This RNA is fully functional at position 45 in the pus1Δ strain. (D–G) Traces 1 (magenta): Point mutations in the 3′ side of the yeast-optimized U2-Ψ45 pseudouridylation pocket affect the corresponding modification activity, especially in the presence of U2-Ψ44 in a “wild-type” yeast strain. Traces 2 (blue): The U2-Ψ45 modification activity is evident in the absence of U2-Ψ44 in the pus1Δ yeast strain. Stars indicate guide RNA-induced pseudouridines.
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FIGURE 6. Prior pseudouridylation of U2 snRNA at position 43 inhibits positioning of Ψ44 by mouse SCARNA8 and S. pombe pugU2-43/44, SPNCRNA.1709. Fluorescent primer extension reactions. (A) Mouse SCARNA8 was expressed in the S. cerevisiae strains pus1Δ (top magenta trace) and pus7Δ (bottom green trace) from a plasmid with a weak CYC1 promoter that allowed prior modification of position 44 in yeast U2 snRNA by endogenous Pus1p. In the pus7Δ strain, modification of position 45 was reduced relative to that in the pus1Δ strain (arrow shows reduction in peak height). Stars indicate SCARNA8-induced pseudouridylation. Modification of position 35 in the pus7Δ strain served as an internal positive control. (B) Top trace (red): Pseudouridylation pattern of U2 snRNA in wild-type S. pombe (strain ED666). Bottom trace (blue): Overexpression of pugU2-43/44ΔUmut RNA caused reduction of pseudouridylation at position 44 (arrow shows reduction in peak height).
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FIGURE 7. Prior 2′-O-methylation of a position in the branch point recognition region inhibits pseudouridylation of the following position. Fluorescent primer extension reactions. (A) Trace 3 (black): Expression of Drosophila scaRNA:MeU2-C41 in the wild-type S. cerevisiae strain BY4741 results in 2′-O-methylation of yeast U2 snRNA at position 41. Traces 1 (red) and 2 (blue): Methylation at position 41 results in dramatic reduction of pseudouridylation at position 42 (trace 2) relative to control (trace 1). (B) Traces 1 (red) and 2 (blue): In HeLa cells, overexpression of the methylation guide RNA for position C40 of U2 snRNA (trace 2) leads to a lower level of U2 snRNA pseudouridylation at position 41 than in control cells transfected with an empty vector (trace 1). The arrow in trace 2 shows reduction in peak height.
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FIGURE 8. U85 scaRNA modification activity on U5 snRNA. (A) Predicted interactions of human U85, Xenopus U85, and human U87 scaRNAs with U5 snRNA. Targeted positions are shown in blue. Mutations introduced in antisense elements of Xenopus U85 and human U87 RNAs are color-coded to match colors used in B–E. (B–E) Fluorescent primer extension reactions. (B,D) 2′-O-methylation and (C,E) pseudouridylation of the 5′ loop of vertebrate U5 snRNA (nucleotides 37–57) inserted into U87 RNA and expressed in wild-type yeast cells. Stars indicate peaks that correspond to modifications induced by human U85 (traces 1, red in B–D) and two Xenopus wild-type U85 (traces 2, green and 4, blue in B–D; traces 1, 3 in E), and Xenopus U85 with mutated Cm45 antisense element, U85ΔC45ASE (traces 3, magenta in B–D; trace 2 in E). Arrowheads point to missing Ψ46. Trace 5, black in B, C, and D: Control samples of U87–U5 substrate RNAs expressed alone in yeast cells. In the tested artificial U5 snRNA substrate, U87–U5[37–57], position 43 is pseudouridylated by an unknown endogenous yeast snoRNA (C,E) and positions 41 and 45 are 2′-O-methylated by U87 RNA itself (B). (D) When the antisense element for positioning Um41 and Cm45 was removed from U87, U87mut-U5[37–57], an additional position became 2′-O-methylated by Xenopus U85; human U85 could not induce this modification. Open circles in C and E indicate stops at 2′-O-methylated positions. In the interest of space, only three Xenopus U85 traces are shown in E; the pattern induced by human U85 (not shown) is very similar to that in trace 1 (green) of Xenopus U85(2); the control U87mut-U5[37–57] alone (not shown) looks as expected, with one peak at Ψ43 and no extra peak at Um41. The sequence of U87–U5 substrate RNA and mutated positions are shown at the bottom of E.
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FIGURE 9. Interactions within a heavily modified region of human 18S rRNA. (A) Base-pairing between human 18S rRNA and guide RNAs SNORA24, SNORA28, and SNORD98 assigned for modification of positions 863, 866, and 867, respectively. (B) Predicted interference between Ψ863, Ψ866, and Gm867 (top row) and differentially modified fragments (second and third rows) experimentally detected by classical biochemical techniques (Maden and Wakeman 1988). Modified positions are colored in blue.
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