ELAV multimerizes on conserved AU4-6 motifs important for ewg splicing regulation
ELAV multimerizes on conserved AU4-6 motifs important for ewg splicing regulation
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DOI：
10.1128/mcb.25.17.7580-7591.2005
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发表时间：
2005-09-01
影响因子：
5.3
通讯作者：
White, K
中科院分区：
文献类型：
--
作者：
Soller, M;White, K
ELAV是果蝇神经元中选择性Pre-mRNA处理的基因特异性调节因子。由于ELAV/Hu蛋白优先结合到AU富集区，而AU富集区通常在内含子和非翻译区中丰富，因此目前还不清楚基因特异性是如何实现的。在这里，我们采用了体外生化实验、系统发育比较和体内分析相结合的方法来研究ELAV与EWG最后一个内含子的结合和剪接调控。ELAV的体外结合研究表明，ELAV在EWG结合部位形成多聚体，形成确定的可饱和的复合体。此外，ELAV-RNA复合体的大小和一系列滴定实验表明，ELAV在最后一个EWG内含子的135个核苷酸上形成了一个十二聚体复合体。对ELAV结合和复合体形成的底物RNA要求的分析表明，分布在整个结合位点的一系列AU(4-6)基序是重要的，但不是严格定义的序列元件。AU(4-6)基序的重要性，而不是它们之间的间距，进一步得到了几个黑腹蛇物种亚群进化保守的支持。最后，利用转基因在果蝇神经元中证明，ELAV介导的EWG内含子6剪接需要几个AU(4-6)基序，而在保守的AU(4-6)基序之间引入间隔序列对剪接的影响最小。综上所述，我们的结果表明，ELAV的多聚化和与多个AU(4-6)基序的结合有助于在复杂的细胞环境中识别和处理目标RNA。
ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. Here we used a combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU(4-6) Motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU(4-6) motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes we demonstrate in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU(4-6) motifs and that introduction of spacer sequence between conserved AU(4-6) motifs has a minimal effect on splicing. Collectively, our results suggest that ELAV multimerization and binding to multiple AU(4-6) motifs contribute to target RNA recognition and processing in a complex cellular environment.