RNA splicing regulation during alcohol withdrawal - Project Summary/Abstract One way in which chronic alcohol exposure produces neuronal adaptations is by changing gene expression. These changes can influence alcohol-drinking behavior and may lead to the development of alcohol use disorder (AUD). Additionally, during alcohol withdrawal, gene expression changes can contribute to the development of negative affective states, such as anxiety and depression, which makes it challenging for individuals to stop consuming alcohol. Mounting evidence from many species indicates that chronic a lcohol exposure also leads to alternatively spliced transcripts in different brain regions. Yet the molecular mechanisms by which alcohol alters RNA splicing remains unknown. This K99/R00 award includes a comprehensive career development and research plan based on Dr. Luana Carvalho’s preliminary data showing that withdrawal from chronic alcohol exposure in male rats increases the expression of genes encoding components of the RNA splicing machinery and leads to changes in RNA splicing. My preliminary data shows increased expression of the splicing factor Poly r(C) binding protein (PCBP1) in the hippocampus (HPC) of ethanol withdrawn rats that present with anxiety and depression-like behavior, as well as in the postmortem HPC of subjects diagnosed with AUD. I also found that PCBP1 is implicated in the mis-splicing of the Hapln2 gene, an important regulator of neuronal conductivity in which loss of function could negatively impact neurotransmission in the context of alcohol use. The scientific goal of this K99/R00 is to investigate RNA splicing, with a focus on PCBP1, as a mechanism by which chronic alcohol exposure and withdrawal leads to molecular alterations and contributes to the emergence of negative affective states. I will manipulate PCBP1 expression using viral-mediated gene delivery to test its behavioral relevance during alcohol withdrawal. I will also perform RNA immunoprecipitation with a PCBP1 antibody, followed by next generation sequencing to identify PCBP1-targets. Finally, I will perform full-length transcriptome sequencing to identify the portfolio of alternatively spliced transcripts in the HPC during chronic alcohol exposure and withdrawal. During my K99 phase, I will gain additional technical training in cutting-edge molecular, bioinformatic and statistical approaches. I will also enhance my leadership skills and receive considerable training in grant writing, oral presentations, and ethics that will be crucial to my success as an independent researcher. During the R00 phase, I will apply all training received to continue this project and further explorer PCBP1 targets in the HPC of humans diagnosed with AUD. Collectively, this work will provide insights into the molecular mechanisms underlying alcohol withdrawal-induced changes on RNA splicing and negative affective states, reveal novel targets and testable hypothesis for future functional studies, and facilitate translational research for finding new targets for AUD treatment.
