Fig 1: ExSpeU1s SM25 mutants with defective protein binding.(a) Schematic representation of RNA secondary structures and sequences of SM25 ExSpeU1 mutants. Highlighted modified nucleotides are shown in grey. (b) Expression levels, quality and nuclear distribution of the ExSpeU1 mutant RNAs. Representative northern blot analysis of ExSpeU1 mutants in total and nuclear RNA fractions. RNA was probed for ExSpeU1 SM25 and for U6 and the histograms below each northern blot show the ExSpeU1 SM25 variants' expression levels relative to U6. Values are the mean±s.e.m. of four independent experiments; *P=0.05, **P=0.01, ***P=0.001. (c,d) Purity of total (T) and nuclear (N) fractions assessed by WB using Human Nuclear Matrix Protein p84, tubulin Ab and by EtBr staining of sRNAs loaded on 8% urea– polyacrylamide gel electrophoresis gel. Transfer RNA (tRNA) is missing in the nuclear fraction. (e) Affinity purification of ExSpeU1 SM25 mutants. Nuclear extracts (NE) from Hek293 cells transfected with the indicated constructs were incubated with the SM25 biotinylated oligonucleotide. Affinity-purified snRNPs were analysed by WB using antibodies against U1–70K, U1A and U1C. (f) RNA-IP analysis of SM25 ExSpeU1 variants. Hek293 NE from cells transfected with ExSpeU1 SM25 variants or not trasfected cells were incubated with antibodies against U1A, 70K, U1C and Sm proteins. RNAs purified from the RIP complexes were analysed by northern blotting. Pulled-down complexes were also analysed by northern blotting with U1 wild-type probe (Supplementary Fig. 9).
Fig 2: Protein composition of ExSpeU1s and endogenous U1 snRNP.(a) Schematic representation of U1 snRNA secondary structure and associated proteins along with the RNA oligonucleotides used in affinity purification, RIP and EMSA. (b) Affinity purified CF11, SM25 and FIX9 ExSpeU1s contain 70K, U1A and U1C. Nuclear extracts (NE) from Hek293 cells, transfected with the indicated ExSpeU1s, or not transfected cells were incubated with the corresponding biotinylated 2'-O-methyl-RNA oligonucleotides. Affinity-purified snRNPs were analysed by western blotting using antibodies against U1–70K, U1A and U1C. (c) RNA-immunoprecipitation analysis of SM25 ExSpeU1. Hek293 NEs from cells transfected with ExSpeU1 SM25 or not transfected cells were incubated with antibodies against U1A, 70K and U1C. RNAs and proteins purified from the RIP complexes were analysed by northern and western blotting, respectively with the indicated probes/antibodies. Mock Ab corresponds to anti-tubulin. (d) CF11 ExSpeU1 snRNP has the same electrophoretic mobility as normal U1 in EMSA. Radiolabelled RNA oligonucleotides complementary to normal U1 (lanes 1–5) or ExSpeU1 CF11 (lanes 6–10) were incubated with nuclear extracts transfected with ExSpe CF11 or mock (not transfected cells). Addition of the indicated antibodies super-shifted the complexes in U1wt and ExSpe CF11. Control EMSA experiments are shown in Supplementary Fig. 5.
Fig 3: Role of 70K protein and stem loop 4 in severely affected exons.(a) Effect of ExSpeU1 variants on mutant minigenes. Minigenes were co-transfected with the ExSpeU1 variants and the splicing pattern was evaluated by RT–PCR. Percentage of exon inclusion was quantified by ImageJ. Data represent the means±s.d. of three independent experiments performed in duplicate. (b) The histogram shows the mean activity of the ExSpeU1 mutants relative to the corresponding native particles calculated as mean of the four minigene systems. For each ExSpeU1 variant, the difference between the basal and induced exon inclusion was calculated as ??. Values obtained for WT particles were set to 1 and then used to calculate the relative activity of each ExSpeU1 mutant. Data represent the mean±s.e.m. of ExSpeU1 mutants relative activity from four minigene systems (Student's t-test ***P=0.001; NS, not significant). (c) Effect of silencing of U1-specific proteins on native ExSpeU1-induced splicing rescue. The efficacy of siRNA treatments on the endogenous protein levels was verified by WB using antibodies against U1A, 70K, U1C. Tubulin was used for normalization. (d) siRNA-treated and control HeLa cells were co-transfected with minigenes and corresponding ExSpeU1s as indicated. The splicing pattern was evaluated by RT–PCR. Data are shown as means±s.d. of three independent experiments performed in duplicate.
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