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. 2010 May 17;189(4):755–767. doi: 10.1083/jcb.200912011

Figure 3.

Figure 3.

Size estimation of the U3 RNP complex. (A) Comparison of U3 RNP protein profiles. U3 RNA was expressed with MYO4-TAP (lane 1) or U1Ap-GFP-TAP (lane 2). TAP-purified complexes from each cell extract were separated by 4–15% SDS-PAGE and silver stained. (B) Myo4–She3 shifts into a 15S RNP complex upon binding U3 RNA. U3 RNA expression was induced (top) or not induced (bottom) in cells expressing MYO4-1/2TAP, and the cell extracts were used for 1/2TAP-purification. The purified complexes were loaded on 10–50% sucrose gradients, and the collected fractions were analyzed by Western blotting. Myo4 bound to U3 RNA purifies as a complex with a size of 15.03 ± 0.90S (n = 6), whereas cargo-free Myo4 purifies at 7.81 ± 0.45S (n = 3). (C) Sedimentation coefficient values of Myo4 associated with U3 RNA and in soluble form. Size determination of U3 RNP complex and Myo4–She3 was based on TAP-purified complexes from cells expressing MYO4-1/2TAP with or without U3 RNA overexpression. Cells expressing MYO2-1/2TAP were used for Myo2p purification.