Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.

The serotonin 2A receptor is widely implicated in genetic association studies and remains an important drug target for psychiatric, neurological, and cardiovascular conditions. RNA sequencing redefined the architecture of the serotonin 2A receptor gene (HTR2A), revealing novel mRNA transcript isoforms utilizing unannotated untranslated regions of the gene. Expression of these untranslated regions is modulated by common single nucleotide polymorphisms (SNPs), namely rs6311. Previous studies did not fully capture the complexity of the sense- and antisense-encoded transcripts with respect to novel exons in the HTR2A gene locus. Here, we comprehensively catalogued exons and RNA isoforms for both HTR2A and HTR2A-AS1 using RNA-Seq from human prefrontal cortex and multiple mouse tissues. We subsequently tested associations between expression of newfound gene features and common SNPs in humans. We find that the human HTR2A gene spans ~66 kilobases and consists of 7, rather than 4 exons. Furthermore, the revised human HTR2A-AS1 gene spans ~474 kilobases and consists of 18, rather than 3 exons. Three HTR2A exons directly overlap with HTR2A-AS1 exons, suggesting potential for complementary nucleotide interactions. The repertoire of possible mouse Htr2a splice isoforms is remarkably similar to humans and we also find evidence for overlapping sense-antisense transcripts in the same relative positions as the human transcripts. rs6311 and SNPs in high linkage disequilibrium are associated with HTR2A-AS1 expression, in addition to previously described associations with expression of the extended 5’ untranslated region of HTR2A. Our proposed HTR2A and HTR2A-AS1 gene structures dramatically differ from current annotations, now including overlapping exons on the sense and anti-sense strands. We also find orthologous transcript isoforms expressed in mice, providing opportunities to elucidate the biological roles of the human isoforms using a model system. Associations between rs6311 and expression of HTR2A and HTR2A-AS1 suggest this polymorphism is capable of modulating the expression of the sense or antisense transcripts. Still unclear is whether these SNPs act directly on the expression of the sense or antisense transcripts and whether overlapping exons are capable of interacting through complimentary base-pairing. Additional studies are necessary to determine the extent and nature of interactions between the SNPs and the transcripts prior to interpreting these findings in the context of phenotypes associated with HTR2A.

The SH-SY5Y neuroblastoma cells are a widely used in vitro model approximating neurons for testing the target engagement of therapeutics designed for neurodegenerative diseases and pain disorders. However, their potential as a model for receptor-mediated delivery and uptake of novel modalities, such as antibody-drug conjugates, remains understudied. Investigation of the SH-SY5Y cell surfaceome will aid in greater in vitro to in vivo correlation of delivery and uptake, thereby accelerating drug discovery. So far, the majority of studies have focused on total cell proteomics from undifferentiated and differentiated SH-SY5Y cells. While some studies have investigated the expression of specific proteins in neuroblastoma tissue, a global approach for comparison of neuroblastoma cell surfaceome to the brain and dorsal root ganglion (DRG) neurons remains uninvestigated. Furthermore, an isoform-specific evaluation of cell surface proteins expressed on neuroblastoma cells remains unexplored. In this study, we define a bioinformatic workflow for the identification of high-confidence surface proteins expressed on brain and DRG neurons using tissue proteomic and transcriptomic data. We then delineate the SH-SY5Y cell surfaceome by surface proteomics and show that it significantly overlaps with the human brain and DRG neuronal surface proteome. We find that, for 32% of common surface proteins, SH-SY5Y-specific major isoforms are alternatively spliced, maintaining their protein-coding ability, and are predicted to localize to the cell surface. Validation of these isoforms using surface proteomics confirms a SH-SY5Y-specific alternative NRCAM (neuron-glia related cell adhesion molecule) isoform, which is absent in typical brain neurons, but present in neuroblastomas, making it a receptor of interest for neuroblastoma-specific therapeutics.

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume. A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI. We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups. The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.