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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Methods. 2017 Apr 13;125:70–83. doi: 10.1016/j.ymeth.2017.04.005

Fig. 10. The five-spliceosomal U snRNAs are associated with in vivo assembled supraspliceosomes at all splicing stages.

Fig. 10

(A, B) Schemes of AdML minigenes, each expressed from a stable cell line from which specific supraspliceosomes at defined splicing stages were affinity purified using the PP7/PP7CP system [70]. Stable cell lines expressing the respective AdML transcripts lacking tag were used as controls. Open boxes represent exons, lines represent introns, and stem-loops represent the PP7 tag inserted either at the 3’UTR or the intron. (A) Constructs expressing AdML-WT. (B) constructs expressing AdML-Mut, having a mutated 3' splice site, designated “x”. (C, D) The affinity purified splicing complexes of AdML-WT-PP73’UTR are assembled in vivo on supraspliceosomes. (C) Upper panel: Affinity purified splicing complexes assembled on the AdML-WT-PP73’UTR transcript, were fractionated in 10–45% glycerol gradients [37] and RNA extracted from each fraction was analyzed by RT-PCR (the size of the PCR product (nt) is given). Lower panel: Western blot analysis of nuclear supernatants enriched for supraspliceosomes prepared from HeLa cells and fractionated in 10–45% glycerol gradients using antibodies against hnRNP G, which is associated with supraspliceosomes [39]. (D) Upper panel: Gallery of affinity purified splicing complexes assembled on AdML-WT-PP73’UTR transcript, visualized by EM, after negative staining with 1% uranyl acetate. Lower panel: Control, EM of eluted material from the control sample (AdML-WT transcript without the PP7-tag). The bar represents 50 nm. (E) Northern blot analysis with probes directed against the five-spliceosomal U snRNAs was performed on RNA extracted from nuclear supernatants (Nuc. Sup.), and from affinity purified supraspliceosomes (Elution), prepared from HeLa cell-lines expressing the AdML constructs either with the PP7 tag or without it (−), as indicated. AdML-WT supraspliceosomes were found assembled on mature RNA, and AdML Mut supraspliceosomes were found assembled on pre-mRNA [38]. Left, the AdML-WT-PP73' UTR supraspliceosomes; middle, the AdML-Mut-PP73'UTR supraspliceosomes; Right, AdML-Mut-PP7IVS supraspliceosomes. The identity of the U snRNA probes is given on the left. Adapted from Ref [38].