Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany.
Nat Struct Mol Biol. 2010 Feb;17(2):216-21. doi: 10.1038/nsmb.1718. Epub 2010 Jan 31.
Reversible protein phosphorylation has an essential role during pre-mRNA splicing. Here we identify two previously unidentified phosphoproteins in the human spliceosomal B complex, namely the pre-mRNA processing factors PRP6 and PRP31, both components of the U4/U6-U5 tri-small nuclear ribonucleoprotein (snRNP). We provide evidence that PRP6 and PRP31 are directly phosphorylated by human PRP4 kinase (PRP4K) concomitant with their incorporation into B complexes. Immunodepletion and complementation studies with HeLa splicing extracts revealed that active human PRP4K is required for the phosphorylation of PRP6 and PRP31 and for the assembly of stable, functional B complexes. Thus, the phosphorylation of PRP6 and PRP31 is likely to have a key role during spliceosome assembly. Our data provide new insights into the molecular mechanism by which PRP4K contributes to splicing. They further indicate that numerous phosphorylation events contribute to spliceosome assembly and, thus, that splicing can potentially be modulated at multiple regulatory checkpoints.
可逆蛋白磷酸化在 pre-mRNA 剪接过程中起着至关重要的作用。在这里，我们鉴定了人剪接体 B 复合物中的两种先前未鉴定的磷酸化蛋白，即 pre-mRNA 加工因子 PRP6 和 PRP31，它们都是 U4/U6-U5 三小核核糖核蛋白（snRNP）的组成部分。我们提供的证据表明，PRP6 和 PRP31 可被人 PRP4 激酶（PRP4K）直接磷酸化，同时它们被并入 B 复合物中。用 HeLa 剪接提取物进行免疫耗竭和互补研究表明，活性人 PRP4K 是 PRP6 和 PRP31 磷酸化以及稳定、功能性 B 复合物组装所必需的。因此，PRP6 和 PRP31 的磷酸化很可能在剪接体组装过程中起着关键作用。我们的数据为 PRP4K 参与剪接的分子机制提供了新的见解。它们进一步表明，许多磷酸化事件有助于剪接体组装，因此剪接可以在多个调节检查点进行潜在的调节。
