Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA.
Nucleic Acids Res. 2010 Jun;38(11):3760-70. doi: 10.1093/nar/gkq028. Epub 2010 Feb 16.
The Hu RNA-binding protein family consists of four members: HuR/A, HuB, HuC and HuD. HuR expression is widespread. The other three neuron-specific Hu proteins play an important role in neuronal differentiation through modulating multiple processes of RNA metabolism. In the splicing events examined previously, Hu proteins promote skipping of the alternative exons. Here, we report the first example where Hu proteins promote inclusion of an alternative exon, exon 6 of the HuD pre-mRNA. Sequence alignment analysis indicates the presence of several conserved AU-rich sequences both upstream and downstream to this alternatively spliced exon. We generated a human HuD exon 6 mini-gene reporter construct that includes these conserved sequences. Hu protein over-expression led to significantly increased exon 6 inclusion from this reporter and endogenous HuD. Studies using truncated and mutant HuD exon 6 reporters demonstrate that two AU-rich sequences located downstream of exon 6 are important. RNAi knockdown of Hu proteins decreased exon 6 inclusion. An in vitro splicing assay indicates that Hu proteins promote HuD exon 6 inclusion directly at the level of splicing. Our studies demonstrate that Hu proteins can function as splicing enhancers and expand the functional role of Hu proteins as splicing regulators.
Hu RNA 结合蛋白家族由四个成员组成：HuR/A、HuB、HuC 和 HuD。HuR 的表达广泛存在。其他三种神经元特异性 Hu 蛋白通过调节 RNA 代谢的多个过程，在神经元分化中发挥重要作用。在之前检查的剪接事件中，Hu 蛋白促进了选择性外显子的跳过。在这里，我们报告了第一个 Hu 蛋白促进包含选择性外显子的例子，即 HuD 前体 mRNA 的外显子 6。序列比对分析表明，在这个选择性剪接的外显子上下游存在几个保守的 AU 富含序列。我们生成了一个包含这些保守序列的人 HuD 外显子 6 迷你基因报告构建体。Hu 蛋白过表达导致该报告和内源性 HuD 中外显子 6 的插入显著增加。使用截断和突变的 HuD 外显子 6 报告基因的研究表明，位于外显子 6 下游的两个 AU 富含序列很重要。Hu 蛋白的 RNAi 敲低降低了外显子 6 的包含。体外剪接分析表明，Hu 蛋白直接在剪接水平促进 HuD 外显子 6 的包含。我们的研究表明，Hu 蛋白可以作为剪接增强子发挥作用，并扩展 Hu 蛋白作为剪接调节剂的功能作用。
