Single-Neuron RNA Modification Analysis by Mass Spectrometry: Characterizing RNA Modification Patterns and Dynamics with Single-Cell Resolution.

Abstract

The entire collection of post-transcriptional modifications to RNA, known as the epitranscriptome, has been increasingly recognized as a critical regulatory layer in the cellular translation machinery. However, contemporary methods for the analysis of RNA modifications are limited to the detection of highly abundant modifications in bulk tissue samples, potentially obscuring unique epitranscriptomes of individual cells with population averages. We developed an approach, single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS), that enables the detection and quantification of numerous post-transcriptionally modified nucleosides in single cells. When compared to a conventional RNA extraction approach that does not allow detection of RNA modifications in single cells, SNRMA-MS leverages an optimized sample preparation approach to detect up to 16 RNA modifications in individual neurons from the central nervous system of Aplysia californica. SNRMA-MS revealed that the RNA modification profiles of identified A. californica neurons with different physiological functions were mostly cell specific. However, functionally homologous neurons tended to demonstrate similar modification patterns. Stable isotope labeling with CD3-Met showed significant differences in RNA methylation rates that were dependent on the identity of the modification and the cell, with metacerebral cells (MCCs) displaying the fastest incorporation of CD3 groups into endogenous RNAs. Quantitative SNRMA-MS showed higher intracellular concentrations for 2'-O-methyladenosine and 2'-O-methylcytidine in homologous R2/LPl1 cell pairs than in MCCs. Overall, SNRMA-MS is the first analytical approach capable of simultaneously quantifying numerous RNA modifications in single neurons and revealing cell-specific modification profiles.