mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain

Abstract

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.[Supplemental material is available online at http://www.genome.org. Sequence reads from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE20895. Software is available for download from http://www.th-wildau.de/bioinformatics/wios/ and http://mayoresearch.mayo.edu/ mayo/research/beutler_lab/wios.cfm.]Microarray-based transcriptome studies have provided a productive approach to the discovery of therapeutic targets in neurological disorders. For example, gene expression profiling of the dorsal root ganglion (DRG) in chronic pain (Griffin et al. 2003) identified several altered genes (Costigan et al. 2002), some of which were subsequently shown to be key modulators of pain (Tegeder et al. 2006). Gene expression analysis using microarray technology suffers from well-known limitations including poor sensitivity and dynamic range, a requirement for substantial requisite amounts of RNA, and a limited capacity to identify new transcripts or RNA splice sites. Given these limitations, it is unlikely that microarray analysis can reveal the full extent of transcriptome reprogramming underlying neurological disorders, such as chronic pain.Ultra-high-throughput RNA sequencing has emerged as a revolutionary technology with superior...

Phosphoinositide signaling in somatosensory neurons

“…PLCε RNA can be detected in whole DRG-s, but it is essentially not expressed in DRG neurons (Thakur et al, 2014), thus the most likely isoforms to be activated by Ca 2+ influx in DRG neurons are PLCδs. PLCδ4 is expressed several fold higher in DRG neurons than the other two PLCδ isoforms PLCδ3 and PLCδ1 (Lukacs et al, 2007; Hammer et al, 2010; Gerhold et al, 2013; Thakur et al, 2014). Accordingly, capsaicin-induced desensitization of TRPV1 was reduced in PLCδ4 −/− mice, but the effect was relatively moderate, pointing to the possible contribution of other PLC(δ) isoforms to phosphoinositide depletion.…”

Selective repression of gene expression in neuropathic pain by the neuron-restrictive silencing factor/repressor element-1 silencing transcription (NRSF/REST)

“…While a precise number of affected individuals is difficult to determine given the variable and subjective nature of the symptoms, a recent systematic review of epidemiological studies of neuropathic pain in the general population estimates the population prevalence of pain with neuropathic characteristics between 6.9% and 10% [61]. The mechanisms responsible for the transition from acute to chronic pain following neural injury are not well understood, but this transition is accompanied by significant changes in the transcriptome of both the central and peripheral circuits [1, 19]. These long-term changes in gene expression profiles are mediated, in part, by epigenetic mechanisms that reorganize chromatin structure.…”

Plasticity of DNA methylation in a nerve injury model of pain

“…PNS injury elicits a dynamic genome response in affected cells reflected in the alteration of hundreds of RNA transcripts in the dorsal root ganglion (DRG). [1][2][3] Whether PNS injury leads to epigenetic remodeling on a grand scale has not been determined but candidate gene studies suggest the possibility. [4][5][6][7] Denk and McMahon 8 suggested in a recent review article that "direct evidence that epigenetic mechanisms could be involved in the development and/or maintenance of chronic pain conditions is only just beginning to surface, and [that] the field is in its infancy;" [that] "the currently available data suggest that epigenetic mechanisms may be important contributors to chronic pain states;" and that "descriptive studies, for instance examination of genome-wide .…”