Coprecipitation of snRNAs by Prp45p. Whole yeast cell extract (splicing extract) was prepared from cells of strain YMA45/2 grown in galactose-based medium, producing protA:Prp45p. As a control, extract was prepared from the parental wild-type strain carrying vector pNOPPATAIL, producing a double protein A epitope (protA). Extracts (50 μL) were mixed with an equal volume of precipitation buffer containing either IgG-agarose beads (I; lanes 3, 6, 9), agarose beads without antibody (B; lanes 4, 7), or protein A-Sepharose beads with prebound anti-Prp8p antibodies (C; lanes 5, 8) and incubated at 4°C for 2 h. The beads were washed in buffer containing 150 mM NaCl (or 75 mM where indicated), deproteinized, and the RNAs precipitated. The samples were resuspended in formamide loading buffer, resolved on a 6% (w/v) polyacrylamide gel, electroblotted to a Hybond-N nylon membrane (Amersham), and probed for U1, U2, U4, U5, and U6snRNAs. Total RNA extracted from 20 μL of extract is shown in lanes 1 and 2.
