An Auts2-Containing RNA-Binding Complex Regulates Transcript Expression in Developing Cerebral Cortex

Molecular functions of the intellectual disability syndrome gene AUTS2 remain uncertain. Two mechanisms of Auts2 function have been proposed. One study found that Auts2 modifies Polycomb repressive complex 1 (PRC1) to activate transcription of target neurodevelopmental genes by recruiting EP300, a histone acetyltransferase. A second study found that cytoplasmic AUTS2 regulates cytoskeletal assembly through PREX1 to promote neurite outgrowth and migration. To further investigate Auts2 functions, we screened for AUTS2 interactors in cerebral cortex, and profiled changes of transcript expression in the developing cortex of Auts2 conditional mutant mice. AUTS2 immunoprecipitation experiments identified interactions with proteins involved in pre-mRNA processing, and mRNAs, including those encoding EP300 and PREX1, both bound by AUTS2-containing complexes and dysregulated in Auts2 mutant cortex. We propose that AUTS2 interacts with an RNA-binding complex that directly targets transcripts encoding Auts2 effectors. This mechanism provides a unifying model that accounts for previously disparate hypotheses of AUTS2 functions. 4 Biological replicates of P0.5 C57Bl6/J Frontal Cortex, 4 Bioloigical Replicates of Auts2 Exon 15 Flx/Flx Emx1-cre conditional knockout. 2 Biological Replicates of P0.5 C57BL6/J Cortex split into Auts2 C-terminal Antibody RIP-Seq, IgG IP and 10% Input (6 samples total in matched trios)智力障碍综合征相关基因AUTS2的分子功能目前仍不明确。目前已提出两种关于Auts2功能的作用机制：一项研究发现，Auts2可通过招募组蛋白乙酰转移酶EP300，修饰多梳抑制复合体1（Polycomb repressive complex 1，PRC1），从而激活靶神经发育基因的转录；另一项研究则显示，细胞质中的AUTS2可通过PREX1调控细胞骨架组装，以促进神经突向外生长与迁移。为进一步探究Auts2的功能，我们在大脑皮层中筛选了AUTS2的互作蛋白，并对Auts2条件突变小鼠发育皮层的转录本表达变化进行了分析。通过AUTS2免疫沉淀实验，我们鉴定到其可与参与前体mRNA（pre-mRNA）加工的蛋白结合，同时还结合了包括EP300和PREX1编码转录本在内的mRNA；这些转录本既会被含AUTS2的复合物所结合，也在Auts2突变小鼠的皮层中出现了表达失调。我们提出，AUTS2可与一种RNA结合复合物互作，该复合物可直接靶向编码Auts2效应蛋白的转录本。这一机制构建了统一的模型，能够解释此前关于AUTS2功能的两种相悖假说。本研究的实验样本包括：①4份P0.5天C57BL/6J小鼠额叶皮层生物学重复样本；②4份Auts2外显子15 Flox/Flx; Emx1-cre条件性敲除小鼠的生物学重复样本；③将P0.5天C57BL/6J小鼠皮层分为3组的2份生物学重复样本，分别为AUTS2 C端抗体RNA免疫沉淀测序（RIP-Seq）组、IgG免疫沉淀（IP）组以及10%输入组，共计6份样本，分为3组匹配的三重重复。