The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA
The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA
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DOI：
10.1101/gad.903101
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发表时间：
2001-10-01
影响因子：
10.5
通讯作者：
White, K
中科院分区：
文献类型：
--
作者：
Lisbin, MJ;Qiu, J;White, K
果蝇神经特异性蛋白ELAV已被证明可以调节神经胶质（nrg）、直立翼和犰狳三种基因的神经特异性剪接。nrg转录物的选择性剪接涉及3'端外显子的选择性包含。在这里，使用一个迷你基因报告基因，我们发现nrg选择性剪接内含子（nASI）具有重建内源性nrg转录物所见的适当神经特异性RNA加工所需的所有决定因素，包括ELAV的调节。体外紫外交联实验显示，来自核提取物的ELAV沿着3200核苷酸长的nASI交联到四个不同的位点；一个EXS位于默认3'剪接位点的聚嘧啶通道。ELAV交联位点（EXSs）具有共同的长链(U)-富序列，而不是精确的共识；每束至少有2个8/10U元件；突变基因报告分析证实了它们的重要性。此外，我们提出了基于四个exs的ELAV靶序列识别标准，这些exs是nASI中(U)丰富但不与ELAV交联的位点，并通过与果蝇（Drosophila virilis nASI）的系统发育比较预测了exs。这些结果表明，ELAV通过与nASI直接相互作用调控nrg选择性剪接。
Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.