Analysis of micrococcal nuclease-resistant regions in U2 snRNA in the 12S, 15S, and 17S U2 snRNPs. (A) In vitro reconstitution of the 15S and 17S U2 snRNPs. The indicated amounts (in microliters) of nuclear extract (NE), SF3a, SF3b, and the 12S U2 snRNP were incubated with 5′ endlabeled oligonucleotide U2A and separated in a 4% polyacrylamide gel. The position of the 15S and 17S U2 snRNPs is indicated on the right. (B) Secondary structure of mammalian U2 snRNA (according to Ares and Igel, 1990; Hartshorne and Agabian, 1990). The Sm-binding site is underlined, the branch site interaction sequence is shown in bold, and the stem-loops are indicated by roman numerals. Sequences complementary to oligonucleotides are shown by thick lines. (C) Analysis of protected fragments of U2 snRNA in the 12S, 15S, and 17S particles by Northern blotting. The 12S U2 snRNP (Mono Q) and reconstituted 15S and 17S U2 snRNPs were incubated in the absence (−) or presence (+) of micrococcal nuclease for 10 min at 30°C. RNA was isolated, separated in a 14% polyacrylamide/8.3 M urea gel, and blotted onto a nylon membrane, followed by detection with oligonucleotides complementary to different portions of U2 snRNA as indicated above each panel. The migration of DNA length markers is indicated on the left of the figure. Please note that the DNA fragments migrate faster than the RNA fragments. Protected U2 snRNA fragments are indicated by lines on the right side of each panel.

Separation of different forms of U2 snRNP by Mono Q chromatography. (A) RNA from 20-μl aliquots of HeLa cell nuclear extract and Mono Q fractions was isolated by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation, separated in a 10% polyacrylamide/8.3 M urea gel, and stained with ethidium bromide. snRNAs and 5S RNA are indicated on the right. (B) Aliquots (5 μl) of HeLa cell nuclear extract (NE), glycerol gradient–purified 17S and 12S U2 snRNPs, and Mono Q fractions were incubated with 5′ endlabeled oligonucleotide U2A (see Fig. 4 B) and separated in a 4% polyacrylamide gel (Brosi et al., 1993a). The different forms of U2 snRNP are indicated on the right.

Analysis of glycerol gradient–purified 15S U2 snRNP. The 15S U2 snRNP (Mono Q) was sedimented through a 15–40% glycerol gradient. (A) RNA from individual gradient fractions was separated in a 10% polyacrylamide/8.3 M urea gel and stained with ethidium bromide. snRNAs are indicated on the left. (B) Proteins were separated in a 10% SDS polyacrylamide gel and stained with silver. The SF3b subunits are indicated by arrows. (C) The 12S U2 snRNP (Mono Q) and SF3b (Mono S), the 15S U2 snRNP (Mono Q; IP), and glycerol gradient fractions of the 15S U2 snRNP were analyzed for presplicing complex formation in the presence of SF1, SF3a, U2AF, and U1 snRNP (see Materials and Methods). A control reaction with nuclear extract (NE) is shown in the second lane. The migration of pre-mRNA and presplicing complex A is indicated on the left. (D) Glycerol gradient–purified SF3b and 15S U2 snRNP were separated in a 10% SDS polyacrylamide gel. Arrows indicate the SF3b subunits. The U2-specific proteins A′ and B′′ are indicated by lines; size markers are shown on the left.

Immunoprecipitation analysis of interactions between SF3a, SF3b, and the 12S U2 snRNP. Combinations of fractions containing SF3a (Mono S), SF3b (Mono S), and the 12S U2 snRNP (Mono Q) were incubated for 15 min at 30°C (Brosi et al., 1993a) followed by precipitation with mAb66 bound to PAS. (A) Aliquots of untreated components or material bound to PAS were separated in a 10% SDS polyacrylamide gel and stained with silver. Lanes 1–3, input fractions of SF3a, SF3b, and the 12S U2 snRNP (representing one-third of the material used in the immunoprecipitations). Lane 4, control reaction of reconstituted 17S U2 snRNP bound to PAS in the absence of mAb66. Lanes 5–12, immunoprecipitation in the presence of the fractions indicated. Lane 13, the 12S U2 snRNP was treated with micrococcal nuclease (MN) before incubation with SF3a and SF3b. Lane 14, SF3b and the 12S U2 snRNP were incubated for 15 min at 30°C, and treated with micrococcal nuclease followed by addition of SF3a and immunoprecipitation. Lane 15, SF3a, SF3b, and the 12S U2 snRNP were incubated for 15 min at 30°C, and treated with micrococcal nuclease followed by immunoprecipitation with mAb66-PAS. Lane 16, control immunoprecipitation of endogenous 17S U2 snRNP. Lane 17, glycerol gradient–purified 15S U2 snRNP. The SF3a and SF3b subunits are indicated on the right of the figure by arrowheads and arrows, respectively. The protein indicated by H represents the heavy chain of mAb66 IgG and two proteins indicated by closed circles are contaminants present in the mAb66 preparation. Protein size standards are shown on the left. (B and C) Aliquots of untreated components or material bound to PAS were separated in a 13% SDS polyacrylamide gel, blotted to nitrocellulose and incubated with mAb4G3 (anti-B′′, panel B) or mAbY12 (anti-Sm, panel C; only the staining of the Sm proteins B and B′ is shown). The migration of U2B′′, Sm proteins B and B′, and the mAb66 light chain (L) is indicated. (D) RNA from HeLa cell nuclear extract (NE), the 12S U2 snRNP, the 12S U2 snRNP treated with micrococcal nuclease (MN-12S), and from the material bound to PAS was separated in a 14% polyacrylamide/8.3 M urea gel and blotted to a nylon membrane. U2 snRNA was detected with radiolabeled antisense U2 snRNA.

General view of the morphology of the 12S, 15S, and 17S U2 snRNPs and SF3b. Preparations of U2 snRNP particles and SF3b were negatively contrasted with uranyl formate and viewed by electron microscopy. (A) 17S U2 snRNP. Arrowheads indicate the filamentous connection between the two globular domains of the 17S U2 snRNP. (B) 12S U2 snRNP. Large arrows point to the core domain of the 12S U2 snRNP and small arrows point to the additional domain containing the A′ and B′′ proteins. (C) 15S U2 snRNP. (D) SF3b. Images of the 12S and 17S U2 snRNPs were taken from previous studies (Kastner et al., 1990; Behrens et al., 1993b).

Characteristic images of SF3b and the 15S U2 snRNP. (A) Each panel (I–VII) shows four examples of the seven principal forms of SF3b with interpretative sketches. The arrows in images of form III indicate a peripheral filamentous structure that can only be observed in this type of projection. (B) Panels I–VII show three examples each of the 15S U2 snRNP with characteristic views of SF3b. The electron micrographs are oriented such that they are comparable to those of SF3b shown in A (except for form II). The arrows in images of form III indicate the filamentous structure that is typical for this type of projection. Interpretative sketches of the 15S U2 snRNPs are shown below the images. These sketches are compared with those of SF3b (A); the shaded area indicates the expected position of the 12S U2 snRNP domain. (C) Images of the 15S U2 snRNP. Panels I–VI show images of the 15S U2 snRNP in which the characteristic morphology of the 12S U2 snRNP is visible. For comparison, panel VII shows two images of the 12S U2 snRNP. The roughly round core domain of the 12S U2 snRNP with the central dot (visible only in some images) is oriented close to the SF3b domain in the 15S U2 snRNP and the domain containing A′ and B′′ is oriented upwards. Interpretative sketches are shown below the images. Bar, 20 nm.

Interpretation of the structure of the 15S and 17S U2 snRNP. (A and D) Electron micrographs of representative 15S and 17S U2 snRNPs with a well visible connection between the two domains. (B and E) Interpretative sketches of the 15S and 17S U2 snRNP images. (C and F) Models of the architecture of the 15S and 17S U2 snRNPs indicating the localization of the SF3b protein complex, the 12S U2 snRNP core, and A′/B′′ domains, and SF3a in the 17S U2 snRNP particle. In addition, a projection of the structure of the U2 snRNA in the RNP is shown. Sm-site, binding site of the core proteins; bp-site, region complementary to the intron branch site; cap, tri-methylated cap structure of U2 snRNA. Bar, 20 nm.