Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign; Department of Chemistry, University of Illinois Urbana-Champaign.
Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign; Department of Chemistry, University of Illinois Urbana-Champaign;
J Vis Exp. 2022 Apr 21(182). doi: 10.3791/63940.
Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described-single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)-to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.
RNA 的转录后修饰 (PTMs) 是参与中枢神经系统 (CNS) 翻译调控的一个研究不足的机制。最近的证据将特定的神经元 RNA 修饰与学习和记忆范式联系起来。不幸的是，用于检测这些表观转录特征的常规方法只能对大量组织中的高度丰富的 RNA 修饰进行特征描述，从而排除了对激活行为回路中单个神经元可能存在的独特 PTM 谱的评估。在本方案中，描述了一种方法——通过质谱法进行单个神经元 RNA 修饰分析 (SNRMA-MS)——以同时检测和定量单个神经元中的许多修饰核糖核苷。该方法使用海洋软体动物加利福尼亚海兔的单个神经元进行验证，从主要 CNS 神经节的手术分离和酶处理开始，以暴露神经元胞体，然后使用锋利的针和微吸管进行手动单个神经元分离。接下来，通过在小体积缓冲液中对样品进行机械和热处理，从单个细胞中释放 RNA，以便随后进行 RNA 消化。然后使用优化的液相色谱-质谱法来鉴定和定量修饰核苷。SNRMA-MS 用于建立具有已知形态和功能的 A. californica 单个鉴定神经元的 RNA 修饰模式。呈现了定性和定量 SNRMA-MS 的示例，突出了 RNA 修饰在神经元网络中单个神经元中的异质分布。
