Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity

Abstract

Despite the advances in our understanding of transcriptome, regulation and function of its non-coding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN), a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN) has a key role in learning and long-term memory storage in Aplysia. We have now identified NAT-SRN in the central nervous system (CNS) and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro-dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduction in levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.

Keywords: Aplysia; aging; antisense RNA; memory; neural circuitry; nocoding RNA.

Figure 1

Identification of natural antisense transcripts of neuropeptide sensorin. Arrows indicate the direction of transcription. (A) The psc1 gene (EST X56770) coding for sensorin mRNA has four exons spanning ~40 KB on the scaffold 926 of Aplysia. AS1-4 represents the locations of qPCR amplicons that are used to detect AS transcript. (B) Northern blot detection of SRN and NAT-SRN from the total RNA of Aplysia CNS using strand specific ribo-probes. (C) qPCR analysis of SRN and NAT-SRN in RNA samples from Aplysia CNS. Primers that can amplify antisense direction of kinesin heavy chain, kinesin light chain and the primers to amplify intronic region of sensorin were used as negative controls. (D) Schematic diagram showing our strategy to study NAT-SRN in sensory neurons (E) qPCR analysis of SRN and NAT-SRN in the cell body and neurites of sensory neurons. Data was normalized to 18s rRNA levels. Error bars are SEM.

Figure 2

Aging associated changes in expression of NAT-SRN in the cell body of Aplysia sensory neurons. (A) Confocal projection images of sensorin RNA (Green) co-stained with β-tubulin protein (Red), DIC and merged images are shown. A no antibody (Ab) control was used as non-specific hybridization control. Scale bar, 20 μm; (B,C) are the percentage changes of SRN and NAT-SRN distribution in the cell body of DIV4-cultured sensory neurons from young (3 months old) and old (9 months old) groups of Aplysia using fluorescently labeled sense and antisense ribo probes, respectively. RNA in situ hybridization analysis of (D) is the normalized intensity of β-tubulin protein distribution in young and old neurites. Normalized mean fluorescence intensities measured using NIH ImageJ are shown in bar graphs. Error bars are SEM. Student's t-test was used to determine statistical significance. “^*” is p < 0.05.

Figure 3

Aging associated changes in expression of NAT-SRN in the neurites of Aplysia sensory neurons. (A) Confocal projection images of sensorin RNA (Green) co-stained with β-tubulin protein (Red), DIC and merged. (B,C) are the percentage changes of SRN and NAT-SRN distribution in the neurites of DIV4-cultured sensory neurons, respectively. (D) is the normalized intensity of β-tubulin protein distribution in young and old neurites. Image analyses were performed as described in Figure 2.

Figure 4

Forskolin exposure enhances expression of NAT-SRN in Aplysia sensory neurons. Sensory neurons of mature Aplysia were cultured in vitro and were treated with forskolin (50 μ M, 30 min) on DIV 4. (A) Confocal projection images of FISH analysis of sensorin RNA (SRN) (Green) and (D) that of AS-sensorin RNA (NAT-SRN) (Green) co-stained with β-tubulin protein (Red), as well as DIC images are shown. A no antibody control was used as non-specific hybridization control. Scale bar, 20 μm. (B,E) show the change in expression levels of SRN and NAT-SRN in forskolin treated neurons when compared with vehicle treated control neurons in cell body and neuritis, respectively. (C,F) are the normalized intensity of β-tubulin protein levels in control and forskolin treated cell body and neuritis, respectively. Normalized mean fluorescence intensities measured using NIH ImageJ are shown in bar graphs. Error bars are SEM. Student's t-test was used to determine statistical significance. “^*” is p < 0.05, “^**” is p < 0.01.