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. 2019 Dec 10;48(3):1508–1530. doi: 10.1093/nar/gkz1148

Figure 2.

Figure 2.

Purification of semi-recombinant holo U7 snRNP via biotin-tagged histone pre-mRNA. (A) The experimental outline. The seven proteins of U7-specific ring were expressed as three sub-complexes and bound, depending on experiment, to either synthetic or T7-generated U7 snRNA. The in vitro assembled core U7 snRNP was incubated with N-terminal FLASH and the resultant complex bound to mH2a/2m-3′B histone pre-mRNA (or another biotin-tagged pre-mRNA) and immobilized on streptavidin beads (SA) for subsequent incubation with nuclear extract and reconstitution of semi-recombinant holo U7 snRNP. (B, C) Analysis of immobilized holo U7 snRNP by silver staining and Western blotting. Core U7 snRNP bound to 139N FLASH (amino acids 1–139 N-terminally tagged with GST) was incubated with SA beads in the absence or presence of mH2a/2m-3′B histone pre-mRNA and/or mouse nuclear extract. Immobilized proteins and RNAs were separated by 4–12% SDS/polyacrylamide gel electrophoresis and visualized by silver staining (panel B) or Western blotting (panel C). Note that only lanes 3 and 4 from panel B are used for Western blotting. Asterisks in panel B indicate proteins of the nuclear extract that nonspecifically bind to SA beads. (D–F). Semi-recombinant holo U7 snRNP was immobilized on SA beads as described in panels A and B with the exception that the recombinant core U7 snRNP was complexed with Δ51 FLASH (lacking the first 51 amino acids that are not required for processing and a GST tag) and bound to mH2a/5m-5′pcB pre-mRNA (panel D) instead of mH2a/2m-3′B. Formation of the complex was facilitated by addition of recombinant SLBP (amino acids 125–223). The immobilized complex was UV eluted and a fraction of solution analyzed by silver staining (panel E) or mass spectrometry (panel F). Lane 2 contains recombinant core U7 snRNP that was bound to mH2a/5m-5′pcB pre-mRNA but not incubated with the mouse nuclear extract.