Biphasic Liquid Microjunction Extraction for Profiling Neuronal RNA Modifications by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

RNA modifications are emerging as critical players in the spatiotemporal regulation of gene expression. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables the simultaneous quantification of numerous enzymatically modified RNAs in a biological sample, conventional RNA extraction and enzymatic digestion protocols that are employed prior to analysis have precluded the application of this technique for small-volume samples. In this study, a biphasic liquid microjunction (LMJ) extraction system using coaxial capillaries that direct and aspirate extraction solvents onto a ~350 μm-diameter sample spot was developed and applied for the extraction of RNA from individual cell clusters in the central nervous system of the marine mollusk Aplysia californica. To maximize RNA recoveries, optimized extraction solvents consisting of 10% methanol and chloroform were evaluated under dynamic and static extraction conditions. An MS-compatible RNA digestion buffer was developed to minimize the number of sample transfer steps and facilitate the direct enzymatic digestion of extracted RNA within the sample collection tube. Compared to RNA extraction using a conventional phenol-chloroform method, the LMJ-based method provided 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissue and also produced mRNA of sufficient purity for reverse transcription polymerase chain reaction amplification. Using this approach, the expression of RNA-modifying enzymes in a given neuronal cell cluster can be characterized and simultaneously correlated with the LC-MS/MS analysis of RNA modifications within the same subset of neurons.

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

“…Gradient elution and MS were performed, as previously described. 33,34 Data Analysis. Modified nucleosides were identified by comparing MS2 spectra and LC retention characteristics to database values.…”

“…30−32 We recently developed an LC−MS-based method optimized for CNS tissues in the neurobiological model animal, Aplysia californica. 33 Using this approach, we found that a nonassociative learning paradigm in A. californica produced timedependent shifts in neuronal RNA modification profiles across CNS subregions (i.e., major ganglia). 34 These characteristic changes in neuronal RNA modifications contributed to increased synthesis of poly-glutamine proteins and neuron excitability, reinforcing the functional role of multiple RNA modifications in CNS plasticity.…”

“…Liquid chromatography (LC)–mass spectrometry (MS) approaches are ideally suited for simultaneously providing quantitative information on RNA modifications for the entire collection of modified nucleosides in an organism. − We recently developed an LC–MS-based method optimized for CNS tissues in the neurobiological model animal, Aplysia californica . Using this approach, we found that a nonassociative learning paradigm in A.…”

Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

“…This landscape, or “epitranscriptome,” is responsive to external stimuli and is involved in regulating translation during heat shock [ 7 ], oxidative stress [ 8 ], methylation stress [ 9 ], and experience-dependent plasticity in the central nervous system [ 10 , 11 , 12 ]. Dramatic variations in RNA modification identity and abundance have also been characterized in small clusters of cells and even single cells [ 13 , 14 , 15 , 16 ], further driving the demand for methods that are capable of rapidly characterizing and quantifying modified RNAs.…”

Characterization of Neuronal RNA Modifications during Non-associative Learning in Aplysia Reveals Key Roles for tRNAs in Behavioral Sensitization

“…Here, we demonstrate that non-associative learning in Aplysia coincides with characteristic time- and region-dependent rearrangements of neuronal RNA modification profiles. Using a liquid chromatography (LC)–tandem mass spectrometry (MS/MS) approach optimized for RNA modifications, 41 we identified higher levels of 7-methylguanosine (m 7 G) in total RNA extracts as well as 1-methyladenosine (m 1 A) and 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U) in tRNA during behavioral sensitization of the tail-elicited siphon withdrawal reflex (TSWR). Treatment of ganglia with mcm 5 s 2 U-rich tRNA isolated from sensitized donors increased levels of poly-Q proteins and produced electrophysiological hallmarks of increased excitability in neurons.…”