A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli

Circular RNAs are a large class of non-coding RNAs present in all eukaryotic taxa. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains largely unexplored, partly due to the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, a highly abundant circRNA that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the corresponding linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides compelling evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof-of-principle for the regulatory role of circRNAs in neural processes. We experimentally assessed the potential impact of circTulp4 downregulation in the brain by performing a comparative transcriptomic analysis of three brain regions between wild-type and a circTulp4-deficient (CD) mouse model mice: hippocampus, cortex, and cerebellum (n hippocampus= 2 wt, 3 CD; n cortex= 3 wt, 3 CD; n cerebellum= 3 wt, 2 CD).环状RNA（circular RNAs，以下简称circRNA）是一类广泛存在于所有真核生物类群中的非编码RNA大家族。尽管目前已鉴定出数千种环状转录本，但绝大多数的生物学功能仍未得到充分解析，这在一定程度上归咎于缺乏构建功能丧失型动物模型的有效手段。本研究聚焦于circTulp4——一种在大脑及突触区室中高度富集的环状RNA。我们构建了circTulp4缺陷型小鼠模型：通过靶向突变生成circTulp4所需的剪接受体位点，且未干扰对应线性mRNA的表达水平与蛋白丰度，借此开展了全面的表型分析。研究结果表明，circTulp4在调控神经元及大脑生理功能中发挥关键作用，可调节兴奋性神经传递的强度以及对厌恶性刺激的敏感性。本研究提供了强有力的证据，证明环状RNA可调控神经元的生物学相关功能，并在表型的多个层面发挥调节作用，为环状RNA在神经过程中的调控功能确立了原理性验证。我们通过对野生型与circTulp4缺陷型（CD）小鼠的三个脑区——海马体、大脑皮层及小脑——开展对比转录组分析，实验性评估了circTulp4表达下调在大脑中的潜在影响：海马体样本量为野生型2例、缺陷型3例；大脑皮层样本量为野生型3例、缺陷型3例；小脑样本量为野生型3例、缺陷型2例。