Crane Andrés B, Inouye Michiko O, Jetti Suresh K, Littleton J Troy
The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
bioRxiv. 2025 Jul 1:2024.05.17.594696. doi: 10.1101/2024.05.17.594696.
RNA editing is a post-transcriptional source of protein diversity and occurs across the animal kingdom. Given the complete profile of mRNA targets and their editing rate in individual cells is unclear, we analyzed single cell RNA transcriptomes from larval glutamatergic motoneuron subtypes to determine the most highly edited targets and identify single neuron editing rules. From ~15,000 genes encoded in the genome, 316 high confidence A-to-I canonical RNA edit sites were identified, with 60 causing missense amino acid changes predicted to alter proteins regulating membrane excitability, synaptic transmission, or neuronal function. Twenty-seven canonical sites were edited at >90% frequency as observed for editing of mammalian AMPA receptors, including coding edits in the Rdl GABA receptor, the nAChRalpha5 and nAChRalpha6 acetylcholine receptors, the Shab K channel, the NCKX30C Na/K-dependent Ca exchanger and the postsynaptic scaffold Shank. However, most sites were edited at lower levels and generated variable expression of edited and unedited mRNAs, suggesting stochastic editing that may provide a mechanism to fine-tune synaptic function similar to alternative splicing. Among these variably edited targets were proteins with well-known presynaptic functions, including the voltage-gated Ca channel Cacophony, the synaptic vesicle fusion regulator Complexin, the active zone scaffolding proteins RBP and Rim, and the endocytosis regulators Lap/AP180 and Endophilin. Comparison of these editing targets across other publicly available RNAseq datasets identified several sites present exclusively in larval motoneurons, indicating the presence of cell-type and/or developmental-specific editing. Further comparisons confirmed the co-transcriptional nature of canonical editing and revealed editing is largely resistant to changes in neuronal activity, with only a few sites displaying evidence of being activity-regulated. Noncanonical editing was also found to occur in these neurons, including a C-to-U edit that altered an amino acid in the capsid hinge domain of the synaptic plasticity regulator Arc1. Together, these data provide insights into how the RNA editing landscape may alter protein function to modulate the properties of two well-characterized neuronal populations in .
RNA编辑是蛋白质多样性的转录后来源，在整个动物界都有发生。鉴于单个细胞中mRNA靶标的完整图谱及其编辑率尚不清楚，我们分析了幼虫谷氨酸能运动神经元亚型的单细胞RNA转录组，以确定编辑程度最高的靶标并确定单个神经元的编辑规则。从基因组中编码的约15,000个基因中，鉴定出316个高可信度的A-to-I经典RNA编辑位点，其中60个导致错义氨基酸变化，预计会改变调节膜兴奋性、突触传递或神经元功能的蛋白质。27个经典位点的编辑频率>90%，这与哺乳动物AMPA受体的编辑情况类似，包括Rdl GABA受体、nAChRalpha5和nAChRalpha6乙酰胆碱受体、Shab K通道、NCKX30C Na/K依赖性钙交换器以及突触后支架蛋白Shank中的编码编辑。然而，大多数位点的编辑水平较低，产生了编辑和未编辑mRNA的可变表达，表明随机编辑可能提供一种微调突触功能的机制，类似于可变剪接。在这些可变编辑的靶标中，有具有众所周知的突触前功能的蛋白质，包括电压门控钙通道Cacophony、突触小泡融合调节因子Complexin、活性区支架蛋白RBP和Rim，以及内吞作用调节因子Lap/AP180和Endophilin。将这些编辑靶标与其他公开可用的RNAseq数据集进行比较，发现了几个仅存在于幼虫运动神经元中的位点；这表明存在细胞类型和/或发育特异性编辑。进一步的比较证实了经典编辑的共转录性质，并揭示编辑在很大程度上不受神经元活动变化的影响，只有少数位点显示出受活动调节的证据。还发现这些神经元中存在非经典编辑，包括一个C-to-U编辑，该编辑改变了突触可塑性调节因子Arc1衣壳铰链结构域中的一个氨基酸。总之，这些数据为RNA编辑图谱如何改变蛋白质功能以调节两种特征明确的神经元群体的特性提供了见解。
