Figure 3.

Interactions between SRm160/300 and snRNPs in the assembly of dsx splicing complexes. (A) U1 snRNP binds to the dsx pre-mRNA independently of the ESE, whereas the ESE and U1 snRNP are required for the assembly of U2, U4/U6, and U5 snRNPs on the dsx pre-mRNA. Biotinylated dsx pre-mRNAs were incubated in mock-depleted or snRNP-depleted splicing reaction mixtures for 40 min before affinity selection on streptavidin-agarose. RNA recovered from the beads was separated on a denaturing 10% polyacrylamide gel and analyzed by Northern hybridization using riboprobes specific for the five spliceosomal snRNAs. Lanes 3–11 contain RNA recovered after affinity selection with biotinylated dsx pre-mRNAs, and lane 2 contains RNA recovered after a control selection performed in the presence of a nonbiotinylated dsx(GAA)₆ pre-mRNA. Selections were performed using the dsx pre-mRNA with no enhancer sequence (lanes 3, 6, 9), with the (GAA)₃ enhancer (lanes 4, 7, 10), or with the (GAA)₆ enhancer (lanes 5, 8, 11, 12) from splicing reaction mixtures containing “mock-depleted” extract (CtrlΔ, lanes 2–5), U1 snRNP-depleted extract (U1Δ, lanes 6–8), or U2 snRNP-depleted extract (U2Δ, lanes 9–11). The selection in lane 12 was performed from a splicing reaction mixture containing an equal mixture of the U1 and U2 snRNP-depleted extracts. Lane 1 contains RNA recovered directly from nuclear extract, representing approximately 3% of the amount of extract used in each selection. (B) A subpopulation of U2 snRNP associates with SRm160/300 in the absence of exogenous pre-mRNA. RNA recovered after immunoprecipitation with rAb-SRm160 (lane 3) or a corresponding preimmune serum (lane 2) from HeLa nuclear extract (lane 1) was analyzed as in A. The amount of nuclear extract represented in lane 1 corresponds to approximately 5% of the amount used for each immunoprecipitation.