Wang Y, Wagner J D, Guthrie C
Department of Biochemistry, University of California, San Francisco, California 94143-0448, USA.
Curr Biol. 1998 Apr 9;8(8):441-51. doi: 10.1016/s0960-9822(98)70178-2.
During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition and formation of the catalytic core. Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA helices. Proof of this hypothesis has, however, been conspicuously lacking.
Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. This activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate. A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase activity and the ATP-dependent RNA unwinding activity.
Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed RNA structure on the spliceosome. Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determined by other interacting protein factors. The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.
在前体mRNA剪接过程中，剪接体内RNA二级结构的动态重排对于内含子识别和催化核心的形成至关重要。属于RNA依赖性ATP酶的DExD/DExH盒家族的剪接因子被认为在通过解开RNA螺旋来指导这些重排中起核心作用。然而，这一假设的证据明显不足。
Prp16是一种DEAH盒蛋白，在体外剪接的第二步中发挥作用。使用各种RNA双链体作为底物，我们表明Prp16具有ATP依赖性的RNA解旋活性。这种活性与RNA底物的单链或双链区域中的序列无关。Prp16的ATP结合基序附近的一个突变（prp16-1）抑制了RNA依赖性ATP酶活性和ATP依赖性RNA解旋活性。
我们的发现提供了强有力的生化证据，表明Prp16可以破坏剪接体上的双链RNA结构。由于纯化的蛋白质在解开RNA双链体时缺乏序列特异性，Prp16在剪接体中的解旋活性的靶向可能由其他相互作用的蛋白质因子决定。解旋活性的证明也将有助于我们理解Prp16如何控制分支点识别的保真度。
