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

Figure 2.

Figure 2.

Myo4, She3, and She2 are the sole, major components of the U3 RNP complex. (A) Myo4, She3, and She2 co-migrate in velocity sedimentation analysis. TAP-purified U3 RNP complexes were resolved on a 10–50% sucrose gradient, and fractions were collected from the bottom (fraction 1). These fractions were analyzed by SDS-PAGE and silver stained. Positions of protein standards thyroglobulin (19S), catalase (11.3S), aldolase (7.3S), and albumin (4.6S) from parallel gradients are indicated. (B) Myo4, She3, and She2 migrate as a 20S RNP complex. TEV-eluted U3 RNP complexes were analyzed as in A, and Western blots were probed with antibodies as indicated. The sedimentation coefficient of U1Ap-GFP-TAP–bound U3 RNP complex was measured as 20.61 ± 0.56S (n = 3). U1Ap-GFP peaks twice, showing proteins bound to U3 RNP complex and unbound free U1Ap-GFP. (C) RNase dissociates the Myo4–She3–She2 complex. TEV-eluted U3 RNP complexes were treated with 0.3 mg/ml RNaseA and further analyzed along with intact U3 RNP complexes in B. The sedimentation coefficient value of Myo4–She3 is reduced to 7.76 ± 0.18S (n = 2) when not bound to RNA cargo.