The regulation of alternative pre-mRNA splicing, activity-induced splicing and adaptive splicing by DNA methylation

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Liu, Ling
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Many molecular bases for the long-term adaptive changes in cells remain unknown. Alternative splicing contributes dramatically to protein diversity and allows for the fine-tuning of gene functions in response to changing extracellular stimulation. It’s a flexible process that could be spatiotemporally and dynamically regulated in response to various extracellular stimuli by the combined effects of specific RNA elements, cis- and trans-acting factors, epigenetic modifications as well as other regulatory factors. The effect of repeated stimulations on gene expression can often be distinct from that of a single stimulation, like in sustained regular exercises or long-term drug abuse. However, it remains unclear how splice variants are controlled in this process, and whether DNA methylation or MeCP2 (Methyl-CpG Binding Protein 2) regulate this potential process. Here, by a depolarization-splicing system, it shows that while the majority of exons kept their homeostatic levels after repeated stimulations, a small group of synaptic exons are indeed adaptively spliced in a significantly different way from or even opposite to that by a single treatment upon membrane depolarization. For further exploration, I established an exon DNA methylation-splicing reporter assay system to conveniently examine the methylation effect on splicing by mutagenesis. In combination with a pair-wise genome/transcriptome-wide analysis, it shows that the adaptively spliced exons are controlled by DNA methylation. More interestingly, a group of adaptive synaptic exons is aberrantly spliced upon DNA methylation change in rat GH3 pituitary cells or due to mutation of the mC- or splicing factor-binding domains of the MeCP2 in patients with Rett syndrome, an autism spectrum disorder. In this thesis, the data established the role of DNA methylation in the adaptive splicing of synaptic exons in response to repeated stimulations by cellular activities and demonstrated similar changes in MeCP2-mutated Rett syndrome patients, many of whom suffer from progressive epileptic attacks after birth. It showed clearly that repeated rather than a single or short-term abnormal neuronal activity plus epigenetic dysregulation of either DNA methylation or MeCP2 aggravate aberrant splicing of synaptic genes, specifically in the hippocampus of Rett syndrome patients and likely other neurological diseases as well.
adaptive splicing, depolarization, RNA-Seq, whole-genome bisulfite sequencing, exon DNA methylation-splicing assay, synaptic exons, Rett syndrome