Abstract
Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states - and reveal a potential target for the treatment of chronic pain.
Highlights
The precise exon composition of expressed genes defines fundamental features of neuronal function (Fiszbein and Kornblihtt, 2017; Lopez Soto et al, 2019; Ule and Blencowe, 2019)
The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors
We find that cell-specific exon DNA hypomethylation permits binding of CCCTC-binding factor (CTCF), the master regulator of chromatin structure in mammals, which, in turn, controls splicing in noxious heat-sensing nociceptors
Summary
The precise exon composition of expressed genes defines fundamental features of neuronal function (Fiszbein and Kornblihtt, 2017; Lopez Soto et al, 2019; Ule and Blencowe, 2019). It is essential to understand the mechanisms that regulate alternative pre mRNA splicing. This dynamic process regulates exon composition for >95% of multi-exon genes according to cell-type and influenced by development, cellular activity and disease (Furlanis and Scheiffele, 2018). The cell-specific actions of RNA binding proteins are relatively well described; these splicing factors promote or repress spliceosome recruitment to pre mRNAs via cis-elements proximal to intron-exon splice junctions (Vuong et al, 2016). DNA binding proteins and epigenetic modifications have been reported to alter alternative pre mRNA splicing by influencing RNA Polymerase II kinetics or splicing factor recruitment (Luco et al, 2011). Epigenetic factors are not generally considered physiologically important regulators of cell-specific alternative pre mRNA splicing in neurons (except see (Ding et al, 2017)
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