A Long Non-Coding RNA (Lrap) Modulates Brain Gene Expression and Levels of Alcohol Consumption in Rats

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with the phenotype of alcohol consumption. The “hub gene” of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. The genetically modified rats were assessed for alcohol consumption and brain RNA expression levels were evaluated using RNASeq. Measures of alcohol consumption in wild type, heterozygous and knockout rats demonstrated that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. More than 20% of the differentially expressed isoforms exhibited evidence for novel alternative splicing between Lrap+/+ and Lrap-/- rats. The GO category of “Response to Ethanol” emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Overall design: RNA expression levels were examined in 9 male rat whole brain samples. Rats were genetically modified using CRISPR/Cas9 to remove a 618 bp region of the genome that codes for Lrap, a novel potentially long non-coding transcript (Crl:WI-Lrapem1Geh). 3 rats were heterozygous for the knockout, 3 rats were homozygous for the knockout, and 3 were wild type controls (Wistar from Charles River).