Roscigno R F, Garcia-Blanco M A
Department of Molecular Cancer Biology, Levine Science Research Center, Duke University Medical Center, Durham, North Carolina 27710, USA.
RNA. 1995 Sep;1(7):692-706.
Pre-spliceosomes, formed in HeLa nuclear extracts and isolated by sedimentation on glycerol gradients, were chased into spliceosomes, the macromolecular enzyme that catalyzes intron removal. We demonstrate that the pre-spliceosome to spliceosome transition was dependent on ATP hydrolysis and required both a U-rich small nuclear ribonucleoprotein (U snRNP)-containing fraction and a fraction of non-snRNP factors. The active components in the non-snRNP fraction were identified as SR proteins and were purified to apparent homogeneity. Recombinant SR proteins (ASF, SC35, SRp55), as well as gel-purified SR proteins, with the exception of SRp20, were able to restore efficient spliceosome formation. We also demonstrate that the pre-spliceosome to spliceosome transition requires phosphorylated SR proteins. This is the first evidence that SR proteins are required for the pre-spliceosome to spliceosome transition, the step at which the U4/U6.U5 tri-snRNP assembles on the pre-mRNA. The results shown here, together with previous data, suggest U snRNPs require SR proteins as escorts to enter the assembling spliceosome.
在HeLa细胞核提取物中形成并通过甘油梯度沉降分离得到的前体剪接体，会转变为剪接体，即催化内含子去除的大分子酶。我们证明，前体剪接体到剪接体的转变依赖于ATP水解，并且既需要富含U的小核核糖核蛋白（U snRNP）组分，也需要一部分非snRNP因子。非snRNP组分中的活性成分被鉴定为SR蛋白，并被纯化至表观均一。除SRp20外，重组SR蛋白（ASF、SC35、SRp55）以及凝胶纯化的SR蛋白能够恢复高效的剪接体形成。我们还证明，前体剪接体到剪接体的转变需要磷酸化的SR蛋白。这是SR蛋白在前体剪接体到剪接体转变过程中发挥作用的首个证据，在这一过程中，U4/U6·U5三snRNP组装到前体mRNA上。此处所示结果与先前数据共同表明，U snRNP需要SR蛋白作为护送者才能进入组装中的剪接体。
