Lin Chunru, Yang Liuqing, Yang Jenny J, Huang Youliang, Liu Zhi-Ren
Department of Biology, Georgia State University, University Plaza, Atlanta, 30303, USA.
Mol Cell Biol. 2005 Sep;25(17):7484-93. doi: 10.1128/MCB.25.17.7484-7493.2005.
We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.
我们之前已经证明，p68 RNA解旋酶作为一种重要的人类剪接因子，在mRNA前体剪接过程中作用于U1 snRNA和5'剪接位点（5'ss）双链体。为了进一步分析p68在剪接体中的功能，我们构建了两个p68突变体（基序V，RGLD突变为LGLD，以及基序VI，HRIGR突变为HLIGR）。ATP酶和RNA解旋试验表明，这些突变消除了RNA依赖性ATP酶活性和RNA解旋活性。这些突变消除了p68在剪接体中的功能，并且还抑制了U1从5'ss的解离，而这些突变体仍然与U1-5'ss双链体相互作用。有趣的是，无活性的p68突变体并没有阻止前剪接体向后剪接体的转变。数据表明，p68 RNA解旋酶可能会主动解开U1-5'ss双链体。该蛋白可能在U4.U6/U5添加过程中也发挥作用，而这一过程并不需要p68的ATP酶和RNA解旋活性。此外，我们在此提供证据证明p68 RNA解旋酶在体内mRNA前体剪接过程中的功能作用。我们的实验还表明，p68在体内与未剪接而非已剪接的mRNA相互作用。
