Northwestern University Feinberg School of Medicine, 303 E. Superior St., Lurie 6-117, Chicago, IL 60611, USA.
Mol Cell Biol. 2011 May;31(9):1812-21. doi: 10.1128/MCB.01149-10. Epub 2011 Feb 22.
Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5' splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5' splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.
调节 tau 外显子 10 剪接在 tau 病中起着重要作用。调节 tau 选择性剪接的顺式元件之一是 tau 外显子 10 5'剪接位点的茎环结构。调节这种茎环结构的 RNA 解旋酶尚不清楚。我们使用 RNA 亲和下拉-串联质谱法搜索与该茎环区域相互作用的剪接调节剂，并鉴定 DDX5/RNA 解旋酶 p68 为 tau 外显子 10 剪接的激活剂。p68 刺激 tau 外显子 10 包含的活性依赖于 RBM4，一种内含子剪接激活剂。RNase H 切割和 U1 保护试验表明，p68 促进茎环结构的构象变化，从而增加 U1snRNP 与 tau 外显子 10 的 5'剪接位点的接近。本研究报告了第一个与剪接位点的茎环结构相互作用并以解旋酶依赖性方式调节选择性剪接的 RNA 解旋酶。我们的工作揭示了 p68 在调节 tau 外显子 10 剪接中的先前未知功能。此外，我们的实验揭示了两个 tau 外显子 10 剪接激活剂之间的功能相互作用，p68 结合在茎环区域，RBM4 与内含子剪接增强子区域相互作用。
