(A) The primary sequence (including the modified nucleotides) and the secondary structure of Xenopus U2 snRNA. There are a total of 13 pseudouridines and 10 2′-O-methylated residues. The Sm-binding site is indicated by a hatched gray box. The underlined nucleotides are involved in base-pairing interactions with the branch site of pre-mRNA. Sequences in dark gray, medium gray, and light gray are involved in base-pairing interactions with U6 snRNA in the spliceosome, known as U2–U6 helices I, II, and III, respectively. (B) The strategy for constructing various U2 snRNAs. In vitro transcribed U2 snRNAs containing uridines (U2 [U]-unmodified) or uridine analogs (U2 [^5FU, Ψ, or ³²pU] in which the uridines are fully substituted with ^5FU, Ψ, or ³²pU) were subjected to site-specific RNase H cleavage directed by 2′-O-methyl RNA–DNA chimeras. Three fragments were generated, mixed or matched, and ligated together with T4 DNA ligase in the presence of a bridging DNA oligonucleotide (see Materials and Methods). U2-BP-5FU containing 5-fluorouridines in the BSRR was created by ligating together the unmodified 5′ fragment (nucleotides 1–29), the unmodified 3′ fragment (nucleotides 47–191), and the modified synthetic oligonucleotide (AGUG^5FUAG^5FUA^5FUC^5FUG^5FU^5FUCU corresponding to nucleotides 30–46; purchased from Dharmacon). U2-BP-Ψ, which contained pseudouridines in the BSRR, was generated by ligating together the unmodified 5′ fragment (nucleotides 1–33), the unmodified 3′ fragment (nucleotides 47–191), and the modified middle fragment (ΨAGΨAΨCΨGΨΨCΨ, corresponding to nucleotides 34–46). U2-BP-³²pU and U2-5′-³²pU, which contain ³²P-radiolabeled uridines in the BSRR and the 5′ region, respectively, were constructed in the same way except that the branch site recognition fragment or the 5′ fragment was radiolabeled with ³²pU.

U2 RNA containing 5-fluorouridines in the BSRR fails to reconstitute U2 function in splicing. (A) In vitro transcribed U2 RNA (lane 4), cellular U2 RNA (lane 5), or U2 RNA containing 5-fluorouridines only in the BSRR (lane 6) was injected into Xenopus oocytes depleted of endogenous U2. After an overnight reconstitution, uniformly ³²P-labeled adenovirus pre-mRNA substrate was injected, and splicing was analyzed by electrophoresis on an 8 M urea-polyacrylamide gel. In lane 3, U2-depleted oocytes were not supplemented with U2. Lane 2 is a control in which pre-mRNA was directly injected into mock-depleted oocytes. Lane 1 is an uninjected pre-mRNA substrate. Lane M is a size marker of MspI-digested pBR322 DNA. (B) Prior to the injection of ³²P-labeled adenovirus pre-mRNA substrate, total nuclear RNA was recovered from the same number of oocytes used in lanes 2–6 in A, and was extended using 5′-end radiolabeled antisense U2 and U6 oligonucleotides and reverse transcriptase (see Materials and Methods). The oligonucleotides and the primer-extension products are indicated. A rather strong signal migrating just below the U2 band was also observed. We currently do not know the nature of this signal.

U2 RNA lacking pseudouridines in the BSRR is unable to participate in spliceosome assembly. Reconstitution was carried out as in Figure 4C ▶. However, after the injection of radiolabeled pre-mRNA, a shorter incubation time was allowed (10 min as opposed to 1.5 h). Oocyte nuclei were subsequently isolated and broken, and splicing complexes were analyzed on a native 4% polyacrylamide gel. The samples are identical to lanes 1–6 in Figure 4C ▶. The positions of complexes H, A, B, and C (see Konarska 1990 regarding the nature of these complexes) are indicated.

The rate of U2 pseudouridylation is faster in the BSRR than in the 5′-end region. U2 RNA containing ³²pU in the BSRR (U2-BP-³²pU, lanes 1–4) or U2 RNA containing ³²pU in the 5′-end region (U2-5′-³²pU, lanes 5–8) was injected into the cytoplasm of Xenopus oocytes. At different time points (indicated above each lane), total nuclear RNA was isolated, treated with nuclease P1, and analyzed by TLC. Lanes 1 and 5 are controls in which uninjected ³²pU regionally labeled U2 was directly digested with nuclease P1 and analyzed by TLC in parallel. The positions of uridylate and pseudouridylate migration are indicated.

Blockage of U2 pseudouridylation in the BSRR inhibits its splicing activity. (A) In vitro transcribed U2 RNA (lanes 3 and 7, U2-unmodified) or U2 RNA containing 5-fluorouridines in the BSRR (lanes 4 and 8, U2-BP-5FU) was injected into Xenopus oocytes. Subsequently, U2 RNA containing ³²P-uridines in the BSRR (lanes 1–4, U2-BP-³²pU) or in the 5′-end region (lanes 5–8, U2-5′-³²pU) was injected into the oocytes. Total nuclear RNA was recovered, digested with nuclease P1, and analyzed by TLC. Lanes 2 and 6 are controls in which no in vitro transcribed U2 (U2-unmodified) or 5-fluorouridine-containing U2 (U2-BP-5FU) was preinjected. Lanes 1 and 5 contain U2 that was labeled region-specifically prior to injection. The positions of ³²P-radiolabeled uridylate and pseudouridylate are indicated. (B) U2-depleted oocytes (lanes 2–9) were injected with U2 RNA containing 5-fluorouridines in the BSRR (lanes 4–6, U2-BP-5FU) or U2 fully substituted with 5-fluorouridine (lanes 7–9, U2-Full 5FU). Subsequently, in vitro transcribed U2 (lanes 5 and 8, U2-unmodified), cellular U2 (lanes 6 and 9, U2-modified), or water (lanes 4 and 7) was injected. After an overnight reconstitution, ³²P-radiolabeled pre-mRNA (³²P-Ad) was injected, and splicing was assessed on a denaturing gel. Lane 1 is a control in which U2 was not depleted, and no RNA was supplemented. In lane 2, oocytes were depleted of U2, but not supplemented with any RNA. In lane 3, oocytes were depleted of U2, and subsequently supplemented with in vitro transcribed U2. The positions of pre-mRNA, spliced lariat intron, and mRNA are indicated. (C) Experiments were performed exactly as in lanes 1–6 in B except that U2-depleted oocytes in lane 6 were supplemented with U2 RNA in which only the uridines in the BSRR were substituted with pseudouridines (U2-BP-Ψ).

Pseudouridines in the BSRR of U2 are crucial for the formation of functionally active U2 snRNP. First, U2 containing 5-fluorouridines in the BSRR was injected into U2-depleted oocytes. After an overnight incubation, the oocytes were then injected with radiolabeled in vitro transcribed U2 (U2-unmodified, top panel) or U2 containing pseudouridines in the BSRR (U2-BP-Ψ, bottom panel). Then, 16 h later, the nuclei were isolated, broken, and loaded onto 20%–30% glycerol gradients (100 mM KCl). After centrifugation, the 23 fractions (~500 μL each) were collected. RNAs were recovered from each fraction and were resolved on 6% denaturing polyacrylamide gels. The positions of 17S and 12S particles are indicated.