Nervous system-wide profiling of presynaptic mRNAs reveals regulators of associative memory

Abstract

Presynaptic protein synthesis is important in the adult central nervous system; however, the nervous system-wide set of mRNAs localized to presynaptic areas has yet to be identified in any organism. Here we differentially labeled somatic and synaptic compartments in adult C. elegans with fluorescent proteins, and isolated synaptic and somatic regions from the same population of animals. We used this technique to determine the nervous system-wide presynaptic transcriptome by deep sequencing. Analysis of the synaptic transcriptome reveals that synaptic transcripts are predicted to have specialized functions in neurons. Differential expression analysis identified 542 genes enriched in synaptic regions relative to somatic regions, with synaptic functions conserved in higher organisms. We find that mRNAs for pumilio RNA-binding proteins are abundant in synaptic regions, which we confirmed through high-sensitivity in situ hybridization. Presynaptic PUMILIOs regulate associative memory. Our approach enables the identification of new mechanisms that regulate synaptic function and behavior.

Figure 1

Isolation of presynaptic compartments for RNA-seq. (A) Neuronal cell isolation results in fragmentation of cells, where cell bodies (left) can be detected separately from neurites (right). (B) Schematic of the dual-fluorescent protein strategy for isolating synaptic and somatic compartments in C. elegans. (C) Representative confocal images of rab-3p::mCherry; rab-3p::RAB-3::GFP transgenic worms. Neurons in the head (outlined in white) and tail (outlined in gray) show distinct expression of fluorescent proteins. Cell bodies (outlined in white, arrows) express somatic mCherry (red), while nerve ring synapses exclusively express GFP (green, arrows. Colocalization of the two fluorescent proteins (yellow) is also evident. (D) FACS plot displaying ability to isolate synaptic (GFP+, green), somatic (mCherry+, red), and synaptic + somatic (GFP+/mCherry+, blue) compartments using adult neuron cell isolation method. (E,F) Principle components analysis of six mCherry+ (red) somatic and GFP+ (green) synaptic RNA-seq samples performed as part of DESeq2 analysis. (E) Principle component analysis of four synaptic (green) and six somatic (red) RNA-seq samples after removal of outliers. Remaining samples cluster by subcompartment.

Figure 2

Isolation of presynaptic mRNAs reveals specialized neuronal functions. (A,B) GO Analysis was performed on “synapse-expressed” genes. Significant GO terms (padj < 0.05) highlight that synapse-expressed genes(green) are predicted to be specialized neuronal genes in terms of both localization (A) and function (B), enriched for membrane related terms and depleted for nuclear terms, unlike ubiquitous (black) and neuron-expressed genes (red).

Figure 3

Characterization of Synaptic DEGs. (A) Volcano plot of Synaptic DEGs (Green) relative to Somatic (Red) samples. FDR for DEGs = 0.05 (B) Synaptic DEGs significantly overlap with previously identified adult-neuron expressed genes P-values: hypergeometric distributions (C) Mammalian orthologs of C. elegans synaptic DEGs function in synapses (for citations, see Table S4) or have been detected in previous synaptic neuropil (blue) and axonal transcriptomic (grey) datasets. (D) GO analysis of known conserved synaptic DEGs reveals synaptic and axonal functions (orange, grey, magenta), and an enrichment of RNA binding proteins and translational regulators (orange).

Figure 4

puf mRNAs are neuronally and axonally localized, and PUFs regulate associative memory. (A) Isolated C. elegans neurons 3 days in vitro (DIV), from animals expressing either somatic (prab-3::mCherry) and synaptic (prab-3::RAB-3::GFP) markers. Soma are circled and labeled, and neurites are indicated on the images. (B). smFISH using Quasar570-labeled probes against individual puf mRNAs in isolated prab-3::RAB-3::GFP neurons. Soma are circled and labeled, and neurites are indicated on the images. (C–E) puf-3 is not required for learning (C) or short-term memory (D), but is necessary for intermediate-term memory (E). (F–H) puf-5 is not required for learning (F) or short-term memory (G), but is necessary for intermediate-term memory (H). Mean ± SEM. n ≥ 8–10 per RNAi treatment. *p < 0.05, ***p < 0.001. Mean ± SEM. n ≥ 8–10 per genotype.