Problem
According to the CDC, in 2021 an estimated 50 million Americans (over 20% of the adult population) suffered from chronic pain (1). Despite this prevalence, therapeutics for pain management are typically characterized by poor efficacy and high risk of abuse. As a result, new pain therapeutics are needed. Novel therapeutic targets may come from a better understanding of the gene expression patterns associated with chronic pain, or from the identification of genes or gene products that are specifically expressed in the peripheral nervous system, where pain signals are received and processed.
In the last decade, advancements in sequencing techniques have allowed scientists to explore gene expression patterns in the peripheral nervous system. Differences in the transcriptome can be driven by species, sex and clinical phenotype (2,3,4). However, the role of alternative splicing of RNAs in creating diverse transcripts has lacked attention. Alternative splicing produces multiple isoforms and has the potential to produce immense transcriptomic and proteomic complexity. Unfortunately, the standard approach to RNA-sequencing, called short read sequencing, only sequences snippets up to 300 bp and often fails to distinguish between spliced isoforms.
Solution
To address this problem, we used long-read sequencing (LRS) to identify and characterize the splice isoforms expressed in human dorsal root ganglia (hDRGs) from human organ donors. The hDRG transmits pain signals from the peripheral nervous system to the central nervous system. Circular consensus sequencing, a type of LRS, allows for accurate measurement of RNA segments up to 10k bp. Using this technique, we identified 19,547 coding isoforms in hDRG, of which 763 were previously unannotated in the human transcriptome. Furthermore, the majority of these novel isoforms were transcripts of genes most highly expressed in neurons. We found multiple novel isoforms of genes associated with nociception (the perception of noxious stimuli), including ASIC3, MRGPRX1, SPP1 and HNRNPK. Finally, we validated the recent identification of a previously unannotated 27 bp exon in the WNK1 gene exclusively expressed in the DRG and associated with an inherited neuropathy.
Impact
In this study, we used long-read sequencing to characterize alternative splicing of RNA species in human dorsal root ganglia. In doing so, we generated a dataset that will serve as a public toolset and a valuable resource for identifying novel RNA isoforms in the peripheral nervous system. As a proof of concept, we identified several novel splice isoforms of genes previously associated with nociception and disease. We anticipate that this dataset from donors without a history of pain will be an important baseline for comparison to any future datasets derived from donors with a history of neuropathic pain.
The authors are grateful to the organ donors and their families for their gift.
References:
SM Rikard, AE Strahan, KM Schmit, GP Guy Jr.. Chronic Pain Among Adults — United States, 2019–2021. MMWR Morb Mortal Wkly Rep 2023;72:379–385.
RY North, Y. Li, P. Ray, LD Rhines, CE Tatsui, G Rao, CA Johansson, H Zhang, YH Kim, B Zhang, G Dussor, TH Kim, TJ Price, and PM Dougherty. Electrophysiological and transcriptomic correlates of neuropathic pain in human dorsal root ganglion neurons. Brain 2019. 142(5): p. 1215-1226.
PR Ray, S Shiers, JP Caruso, D Tavares-Ferreira, I Sankaranarayanan, ML Uhelski, Y Li, RY North, C Tatsui, G Dussor, MD Burton, PM Dougherty, and TJ Price. RNA profiling of human dorsal root ganglia reveals sex differences in mechanisms promoting neuropathic pain. Brain 2023. 146(2): p. 749-766.
Ray, P., A. Torck, L. Quigley, A. Wangzhou, M. Neiman, C. Rao, T. Lam, J.Y. Kim, T.H. Kim, M.Q. Zhang, G. Dussor, and T.J. Price, Comparative transcriptome profiling of the human and mouse dorsal root ganglia: an RNA-seq-based resource for pain and sensory neuroscience research. Pain, 2018. 159(7): p. 1325-1345.
