|
Abstract:
|
Gamma -aminobutyric acid type B (GABAB ) receptor is a G protein coupled receptor (GPCR ) that mediates decreased neural activity . It has two subunits , GABAB1 and GABAB2 . Previous complementary DNA (cDNA ) microarray data showed strong GABAB1 signals from human prefrontal cortex using an intron 4 region probe , and these studies indicated that novel intron 4 containing GABAB1 splicing variants exist . We cloned GABAB1k , l , m , and n including mouse GABAB1j . Expression of these variants are much lower than other major known splicing variants , but GABAB1k , l , m , and n levels are similar across brain tissues . GABAB1l and GABAB1m impair GABAB receptor induced function . To better define GABAB1 splicing in alcoholic brains , whole transcriptome shortgun sequencing (RNA -seq ) experiments were proposed . Due to the complexity of GABAB1 splicing , we used gene specific libraries as well as whole transcriptome libraries to maximize GABAB1 specific splicing junction search . The splicing junction search data found that GABAB1 gene is 2 to 3 times longer than the previous known gene length . Extremely low expression at 5’ end exons was found , and GABAB1 exons were grouped based on expression levels . Chronic alcohol altered exon /intron expression and splicing junctions more than overall gene expression . Decreased exon expression at a GABA binding site , a transmembrane domain (TM ) , and a microRNA (miRNA ) binding site may diminish the normal GABAB1 transcript population and compromise signal transduction following chronic alcohol exposure . This may explain why GABAB receptor agonists have therapeutic benefit in treating alcoholism . During the sequence mapping , read pile -ups and gaps were found from whole transcriptome libraries in known exons . These may prevent single nucleotide polymorphism (SNP ) and splicing junction identification and gene expression calculations . Sequence analysis found sequence biases from their mapped reads . The major sequence biases were from RNaseIII RNA fragmentation and T4 polynucleotide kinase (T4PNK ) reaction . Heat fragmentation and OptiKinase treatment removed the read pile -ups and gaps including the sequence biases . The identification of RNaseIII target sequences can be incorporated into methods of miRNA gene prediction . These data showed the complexity of GABAB1 receptor splicing and the perturbation of splicing by chronic alcohol abuse demonstrate the power of RNA -seq to provide new insight into gene expression and the role of GABAB receptors in alcoholism . In addition , many other important brain genes may have unexplored splicing variants which will be important for alcoholism and other psychiatric diseases . Also , new RNA -seq library constructions improved the quality of gene expression studies . |