RNA-Seq of neural stem cells from Wild Type (WT) and Armc5 knockout mice. RNA-Seq of neural stem cells from Wild Type (WT) and Armc5 knockout mice

ARMC5 is a protein containing an armadillo domain (ARM) and a BTB domain. Its gene knockout caused many phenotypes, including dwarfism, compromise T-cell immunity, and adrenal gland hypertrophy. ARMC5 mutation in humans is associated with bilateral macronodular adrenal gland hypertrophy. We found that AMC5 KO mice suffered from an increased incidence of neural tube defects (NTDs). We revealed that ARMC5 complexed with CUL3 and POLR2A and was part of a novel POLR2A-specific ubiquitin ligase (E3). This E3 was the dominant DNA damage-independent POLR2A-specific E3 in developing neural tubes and neural precursor cells under a physiological condition. ARMC5 gene knockout (KO) caused diminished POLR2A ubiquitination and compromised POLR2A degradation via proteasomes. Surprisingly, the absence of this E3 did not lead to generalized Pol II stalling and the subsequent generalized decrease of mRNA transcription but caused an enlarged Pol II pool size, which dysregulated 108 genes in NPCs, including some known to neural development. ARMC5 KO in the intestine downregulated FOHL1 expression, which was essential in folate absorption. Whole-exome sequencing of 511 myelomeningocele (MM) patients revealed nine highly deleterious mutations in the ARMC5 coding sequence. A significant deleterious mutation Arg429Cys found in MM patients drastically weakened the interaction between ARMC5 and POLR2A, supporting our hypothesis that such mutations in ARMC5 increased the NTD risks by compromising the POLR2A-specific E3 activity. Our results indicated that this novel ARMC5-CUL3-RBX1 E3 played a critical role in Pol II pool homeostasis, and ARMC5 mutation was a modifier of NTD risks in mice and humans. Overall design: Neural stem cell mRNA profiles from WT and Armc5(-/-) mice

ARMC5是一种包含armadillo结构域（armadillo domain, ARM）与BTB结构域的蛋白质。其基因敲除（gene knockout, KO）可引发多种表型，包括侏儒症、T细胞免疫功能受损及肾上腺增生。人类ARMC5突变与双侧大结节性肾上腺增生密切相关。本研究发现，ARMC5基因敲除（KO）小鼠的神经管缺陷（neural tube defects, NTDs）发生率显著升高。我们揭示，ARMC5可与CUL3及POLR2A形成复合物，属于一类新型的POLR2A特异性泛素连接酶（ubiquitin ligase, E3）。在生理条件下，该E3是发育中的神经管及神经前体细胞中，占主导地位的非DNA损伤依赖性POLR2A特异性E3。ARMC5基因敲除会降低POLR2A的泛素化水平，并损害POLR2A经蛋白酶体的降解过程。令人意外的是，该E3的缺失并未引发广泛的RNA聚合酶II（Pol II）停滞及随后的mRNA转录普遍下调，反而导致Pol II池规模扩大，进而使神经前体细胞（neural precursor cells, NPCs）中的108个基因表达失调，其中包含若干已知与神经发育相关的基因。在肠道中敲除ARMC5会下调FOHL1的表达，而该基因对叶酸吸收至关重要。对511例脊髓脊膜膨出（myelomeningocele, MM）患者开展全外显子测序后，在ARMC5编码序列中发现9个具有高度有害性的突变。在MM患者中检出的有害突变Arg429Cys，可大幅削弱ARMC5与POLR2A之间的相互作用，验证了我们的假说：ARMC5的此类突变会通过损害POLR2A特异性E3的活性，增加神经管缺陷的患病风险。本研究结果表明，这种新型的ARMC5-CUL3-RBX1 E3在Pol II池稳态调控中发挥关键作用，而ARMC5突变是小鼠及人类神经管缺陷风险的修饰因子。整体实验设计：取自野生型（wild type, WT）与Armc5敲除（Armc5(-/-)）小鼠的神经干细胞mRNA表达谱