Bioorthogonal Metabolic Labeling of Nascent RNA in Neurons Improves the Sensitivity of Transcriptome-Wide Profiling

Abstract

Transcriptome-wide expression profiling of neurons has provided important insights into the underlying molecular mechanisms and gene expression patterns that transpire during learning and memory formation. However, there is a paucity of tools for profiling stimulus-induced RNA within specific neuronal cell populations. A bioorthogonal method to chemically label nascent (i.e., newly transcribed) RNA in a cell-type-specific and temporally controlled manner, which is also amenable to bioconjugation via click chemistry, was recently developed and optimized within conventional immortalized cell lines. However, its value within a more fragile and complicated cellular system such as neurons, as well as for transcriptome-wide expression profiling, has yet to be demonstrated. Here, we report the visualization and sequencing of activity-dependent nascent RNA derived from neurons using this labeling method. This work has important implications for improving transcriptome-wide expression profiling and visualization of nascent RNA in neurons, which has the potential to provide valuable insights into the mechanisms underlying neural plasticity, learning, and memory.

Keywords: CuAAC; Nascent RNA; UPRT; neuron; transcriptome-wide profiling.

Figure 1.

Illustrative graph depicting the production of stimulus-induced nascent RNA versus pre-stimulus steady-state RNA over time. Stimulus exposure (i.e. cellular activation) occurs during a set time period indicated by the red dotted lines.

Figure 2.

Overview of cell-type-specific bioorthogonal meta-bolic labeling of nascent RNA in mammalian cells using the UPRT-5EUracil system.

Figure 3.

Robust nascent RNA labeling in neurons using the UPRT-5EUracil system. (A) UPRT is highly overexpressed in mouse PCNs using a synapsin I-driven lentiviral construct. UPRT lentivirus (+), GFP lentivirus (−), un-paired Student’s t-test (α=0.05, n=3) ****P<0.0001. (B) Visualization of alkyne-labeled biotinylated RNA on a dot blot (n=1/condition), which demonstrates effective nascent RNA capture in UPRT+ cells after treatment for 3h with 5EUracil (UPRT+5EUracil). (C) Visualization of nascent RNA labeled with 5EUracil (green) and HA-UPRT(red) in UPRT+ neurons (n=1). Scalebar: 50μm.

Figure 4.

Pipeline for processing RNA from activated (KCl+) and non-activated (KCl-) neurons, which are then subdivided into two different groups for sequencing: input and enriched.

Figure 5.

Nascent RNA sequencing has improved sensitivity compared to standard RNA-seq. A Venn diagram illustrates the overlap of differentially expressed genes (KCl- Vs KCl+) detected using standard RNA-seq (input, left gray circle) and nascent RNA sequencing (enriched, right green circle). The four experimental groups shown are as follows: input KCl-, input KCl+, enriched KCl, enriched KCl+ (4 biological replicates per group). For gene target lists that were specifically identified in either the input or enriched differential gene expression analysis, the FDR value had to be both <0.05 within the selected comparison and >0.1 in the other so as to exclude any targets that were close to being significant in both (overlap).