Alternative splicing is recognized as a major mechanism to increase protein diversity by shuffling coding gene sequences. Genetic mutations that disrupt alternative splicing have been repeatedly identified as risk factors for autism. In extreme cases, one human gene can generate hundreds of different protein isoforms. This remarkable increase of protein complexity plausibly explains the discrepancy between species complexity and gene numbers. Given the critical roles of alternative splicing in autism and enormous cell types uncovered in the neocortex, an important priority for the field is to comprehensively assess cell-type-specific isoforms for high-confidence autism genes during neocortical development.
A recent study by Xiaochang Zhang, Christopher Walsh and colleagues showed that alternative splicing is widespread between neural progenitors and neurons1. Interestingly, they identified that autism-associated mutations in ANK2 are found in the specific splice isoform that is expressed during cortical development, indicating a neuron-specific disease mechanism. They further showed that intronic mutations causing changes in alternative splicing can cause a unique human brain malformation. These results, together with findings from other groups, led to the hypothesis that cell-type-specific mRNA splicing critically regulates autism risk genes and that mutations associated with autism can impact the relative expression levels of different mRNA isoforms.
The primary goal of the current proposal is to apply long-read sequencing approaches to study ‘full-length’ mRNA isoforms and to investigate their dynamic regulation between different cell types in the neocortex. Such knowledge is expected to lead to a better understanding of gene expression and autism pathogenesis. To achieve this goal, Zhang’s group will take advantage of droplet-barcoded single-cell RNA sequencing to identify diverse progenitor and neuronal cell types in the developing mouse neocortex. In parallel, they will identify full-length mRNA isoforms with long-read sequencing. Integration of cell-type information and long reads will enable Zhang and colleagues to identify cell-type-specific full-length splice isoforms. The alternative splicing patterns of autism risk genes will be the primary focus of these analyses.
The second goal of the project is to determine the impact of alternative mRNA splicing on protein expression and to apply this knowledge to investigate causal mutations in autism. In collaboration with Xin He’s lab at the University of Chicago, Zhang and colleagues plan to test this by analyzing cell-type-specific alternative splicing events in autism risk genes, and determining their impact on protein expression. Zhang’s team also plans to pinpoint the effect of pathogenic mutations on splicing isoforms and infer their cell-type-specific impact.
Taken together, results from these studies are expected to identify cell-type-specific isoforms of autism risk genes that are expressed during neocortical development and to uncover isoform-specific functions.