Purification of U3 RNP complex. (A) Schematic representation of constructs used for U3 RNP complex purification. The RNA-binding protein is expressed from the constitutive glycerol phosphate dehydrogenase (GPD) promoter, and contains U1Ap, GFP, TAP, and an NLS. The RNA partner is expressed from the inducible galactose (GAL) promoter, and consists of four repeats of U1A aptamer, the U3 localization element, and an ADH1 terminator. (B) GFP-bound U3 RNA localizes to the bud tip using the modified tagged RNA system. Expression of 4×U1A-tagged U3 RNA was induced in cells containing U1Ap-GFP-TAP, and GFP signals in live cells were observed by fluorescence microscopy. The outline was drawn based on the DIC image. The nuclear GFP signal is from U1Ap-GFP-TAP not bound to RNA. Bar, 10 µm. (C) Silver staining of TAP-purified complexes. Extracts prepared from cells expressing U1Ap-GFP-TAP only (lane 1), U1Ap-GFP-TAP and 4xU1A-tagged U3 RNA (lane2), or U1Ap-GFP-TAP and 4xU1A-tagged ADH2 (77 NT) RNA (lane 3) were used for TAP purification. The purified complexes were separated on a 4–15% gradient SDS-polyacrylamide gel, and silver stained. (D) Myo4, She3, and She2 specifically copurify with U3 RNA. Purified complexes from C were separated by 10% SDS-PAGE in the same order as in C and analyzed by Western blotting. U1Ap-GFP was detected by an anti-GFP antibody. (E) Mass spectrometry analysis of U3 RNA copurifying proteins. TAP-purified U3 RNP complexes were separated by SDS-PAGE and stained with Coomassie blue. Protein bands of interest were excised from gels and identified by mass spectrometry analysis (LC-MS/MS).