ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

Summary from GEO:<br/><br/>"ELAV/Hu factors are conserved RNA binding proteins that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, still little is known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, we find that ectopic expression of the three family members (Elav, Fne and Rbp9) induces overlapping changes to hundreds of cassette exon and dozens of alternative last exon (ALE) splicing events. Reciprocally, double mutants of elav/fne, but not elav alone, have opposite effects on both classes of regulated mRNA processing events in the larval CNS. While manipulation of Drosophila ELAV/Hu factors induces both exon skipping and inclusion, motif analysis indicates their major direct effects are to suppress cassette exon usage. Moreover, the roles of ELAV/HU factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both involve local suppression of proximal polyadenylation signals via ELAV/Hu binding sites downstream of cleavage sites. The phenotypic impact of combined ELAV/Hu activities in neural mRNA processing is overt, since fne loss strongly enhances neuronal differentiation phenotypes in elav mutants. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu proteins orchestrate multiple conserved programs of neuronal mRNA processing by suppressing alternative exons and polyadenylation sites."<br/> <br/>Overall design from GEO:<br/><br/>"RNA-seq profiles from S2R+ cells with the overexpression of wildtype Elav, Rbp9, Fne and RRM mutant versions of Elav, Rbp9, Fne and BrdU-CLIP of wildtype Elav and RRM mutant variant."

摘要自GEO：

“ELAV/Hu因子是一类保守的RNA结合蛋白，其在mRNA处理和调控中扮演着多样的角色。始祖成员果蝇Elav于35年前被确认为一种关键的神经因素。尽管如此，关于其对转录组的影响及其与同源基因的功能重叠，仍所知甚少。基于我们近期的研究发现，神经特异性延长的3' UTR异构体由ELAV/Hu因子共同决定，我们探讨了其对剪接的影响。尽管已知果蝇的剪接靶标仅有少数，但我们发现，三种家族成员（Elav、Fne和Rbp9）的异位表达导致数百个盒式外显子和数十个选择性最末外显子（ALE）剪接事件发生重叠变化。反之，elav/fne的双突变体而非单独的elav，对幼虫中枢神经系统（CNS）中调控的mRNA处理事件的两种类型产生了相反的影响。虽然果蝇ELAV/Hu因子的操纵既可引起外显子跳跃又可引起外显子包含，但模式分析表明，其主要直接效应是抑制盒式外显子的使用。此外，ELAV/HU因子在全局促进神经元中远端ALE剪接中的作用机制与终端3' UTR延伸密切相关，因为它们都涉及通过ELAV/Hu结合位点在切割位点下游对近端多聚腺苷酸化信号的本地抑制。ELAV/Hu在神经mRNA处理中的表型影响显著，因为fne的缺失显著增强了elav突变体中的神经元分化表型。最终，我们提供了哺乳动物神经元中保守性的证据，这些神经元经历广泛的远端ALE和APA长度延伸程序，与调控多聚腺苷酸化位点下游的ELAV/Hu基序相关。总体而言，ELAV/Hu蛋白通过抑制选择性外显子和多聚腺苷酸化位点，协调了神经元mRNA处理的多个保守程序。”

GEO的总体设计：

“来自S2R+细胞的RNA测序数据，包括野生型Elav、Rbp9、Fne和Elav、Rbp9、Fne的RRM突变体，以及野生型Elav和RRM突变体的BrdU-CLIP。”