Soller Matthias, White Kalpana
Department of Biology, Brandeis University, Waltham, MA 02454, USA.
Mol Cell Biol. 2005 Sep;25(17):7580-91. doi: 10.1128/MCB.25.17.7580-7591.2005.
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.
ELAV是果蝇神经元中前体mRNA可变加工的基因特异性调节因子。由于ELAV/Hu蛋白优先结合富含AU的区域，这些区域通常在内含子和非翻译区中大量存在，因此尚不清楚基因特异性是如何实现的。在这里，我们结合体外生化实验、系统发育比较以及果蝇转基因的体内分析，来研究ELAV与ewg基因最后一个内含子的结合及剪接调控。ELAV的体外结合研究表明，ELAV在ewg结合位点上多聚化并形成一个明确且可饱和的复合物。此外，对ELAV-RNA复合物的大小分析和一系列滴定实验表明，ELAV在ewg基因最后一个内含子的135个核苷酸上形成十二聚体复合物。对ELAV结合和复合物形成的底物RNA需求分析表明，分布在整个结合位点上的一系列AU(4-6)基序很重要，但不是严格定义的序列元件。几个黑腹果蝇物种亚组中的进化保守性进一步支持了AU(4-6)基序而非它们之间的间隔的重要性。最后，我们利用转基因在果蝇神经元中证明，ELAV介导的ewg基因内含子6剪接调控需要几个AU(4-6)基序，并且在保守的AU(4-6)基序之间引入间隔序列对剪接的影响最小。总体而言，我们的结果表明，ELAV多聚化以及与多个AU(4-6)基序的结合有助于在复杂的细胞环境中识别和加工靶RNA。
