Figure 5: 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).