Splicing-specific transcriptome-wide association uncovers genetic mechanisms for schizophrenia.

Recent studies have highlighted the essential role of RNA splicing, a key mechanism of alternative RNA processing, in establishing connections between genetic variations and disease. Genetic loci influencing RNA splicing variations show considerable influence on complex traits, possibly surpassing those affecting total gene expression. Dysregulated RNA splicing has emerged as a major potential contributor to neurological and psychiatric disorders, likely due to the exceptionally high prevalence of alternatively spliced genes in the human brain. Nevertheless, establishing direct associations between genetically altered splicing and complex traits has remained an enduring challenge. We introduce Spliced-Transcriptome-Wide Associations (SpliTWAS) to integrate alternative splicing information with genome-wide association studies to pinpoint genes linked to traits through exon splicing events. We applied SpliTWAS to two schizophrenia (SCZ) RNA-sequencing datasets, BrainGVEX and CommonMind, revealing 137 and 88 trait-associated exons (in 84 and 67 genes), respectively. Enriched biological functions in the associated gene sets converged on neuronal function and development, immune cell activation, and cellular transport, which are highly relevant to SCZ. SpliTWAS variants impacted RNA-binding protein binding sites, revealing potential disruption of RNA-protein interactions affecting splicing. We extended the probabilistic fine-mapping method FOCUS to the exon level, identifying 36 genes and 48 exons as putatively causal for SCZ. We highlight VPS45 and APOPT1, where splicing of specific exons was associated with disease risk, eluding detection by conventional gene expression analysis. Collectively, this study supports the substantial role of alternative splicing in shaping the genetic basis of SCZ, providing a valuable approach for future investigations in this area.

Long-read RNA-seq demarcates cis- and trans-directed alternative RNA splicing.

Genetic regulation of alternative splicing constitutes an important link between genetic variation and disease. Nonetheless, RNA splicing is regulated by both cis-acting elements and trans-acting splicing factors. Determining splicing events that are directed primarily by the cis- or trans-acting mechanisms will greatly inform our understanding of the genetic basis of disease. Here, we show that long-read RNA-seq, combined with our new method isoLASER, enables a clear segregation of cis- and trans-directed splicing events for individual samples. The genetic linkage of splicing is largely individual-specific, in stark contrast to the tissue-specific pattern of splicing profiles. Analysis of long-read RNA-seq data from human and mouse revealed thousands of cis-directed splicing events susceptible to genetic regulation. We highlight such events in the HLA genes whose analysis was challenging with short-read data. We also highlight novel cis-directed splicing events in Alzheimer's disease-relevant genes such as MAPT and BIN1. Together, the clear demarcation of cis- and trans-directed splicing paves ways for future studies of the genetic basis of disease.

Massively parallel screen uncovers many rare 3' UTR variants regulating mRNA abundance of cancer driver genes.

Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3' UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.

Widespread RNA hypoediting in schizophrenia and its relevance to mitochondrial function.

RNA editing, the endogenous modification of nucleic acids, is known to be altered in genes with important neurological function in schizophrenia (SCZ). However, the global profile and molecular functions of disease-associated RNA editing remain unclear. Here, we analyzed RNA editing in postmortem brains of four SCZ cohorts and uncovered a significant and reproducible trend of hypoediting in patients of European descent. We report a set of SCZ-associated editing sites via WGCNA analysis, shared across cohorts. Using massively parallel reporter assays and bioinformatic analyses, we observed that differential 3' untranslated region (3'UTR) editing sites affecting host gene expression were enriched for mitochondrial processes. Furthermore, we characterized the impact of two recoding sites in the mitofusin 1 (MFN1) gene and showed their functional relevance to mitochondrial fusion and cellular apoptosis. Our study reveals a global reduction of editing in SCZ and a compelling link between editing and mitochondrial function in the disease.

Allele-specific alternative splicing and its functional genetic variants in human tissues.

Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale data sets of the Genotype-Tissue Expression (GTEx) project to study genetically modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues versus individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMAS-associated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.