Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms

Abstract

Human brain development is under tight molecular genetic control and the recent advent of single-cell genomics has revolutionized our ability to elucidate the diverse underlying cell-types and states. Although RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, previous work has not systematically investigated the role of cell-type-specific splicing or transcript-isoform diversity during human brain development. Here, we leverage single molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identify 214,516 unique isoforms, corresponding to 22,391 genes. Remarkably, we find that 72.6% of these are novel and together with >7,000 novel-spliced exons expands the proteome by 92,422 proteoforms. We uncover myriad novel isoform switches during cortical neurogenesis, implicating previously-uncharacterized RNA-binding protein-mediated and other regulatory mechanisms in cellular identity and disease. Early-stage excitatory neurons exhibit the greatest isoform diversity and isoform-based single-cell clustering identifies previously uncharacterized cell states. Leveraging this resource, we re-prioritize thousands of rare de novo risk variants associated with neurodevelopmental disorders (NDDs) and reveal that risk genes are strongly associated with the number of unique isoforms observed per gene. Altogether, this work uncovers a substantial contribution of transcript-isoform diversity in cellular identity in the developing neocortex, elucidates novel genetic risk mechanisms for neurodevelopmental and neuropsychiatric disorders, and provides a comprehensive isoform-centric gene annotation for the developing human brain.

Long-read RNA sequencing identifies region- and sex-specific C57BL/6J mouse brain mRNA isoform expression and usage

“…Another future direction includes investigating classes of transcript diversity and structure (i.e., promoter usage and 3′ end choice) as done in ENCODE4 ( 49), but with an emphasis on studying differences across sexes in the brain. There is also a need to investigate sex differences in splicing across the lifespan, including early development (50,51) and aging (38). Finally, future research could combine long-read transcriptomics with measures of neuronal activity to discern the effects of AS on signal transmission across sexes (52).…”

Transcriptional Determinism and Stochasticity Contribute to the Complexity of Autism AssociatedSHANKFamily Genes

“…We noted that the longest annotated SHANK3/Shank3 transcripts in humans (NM_001372044.2, 7,691 bp, hg38) and mice (NM_021423.4, 7,380 bp, mm39) have not been detected in any published long-read RNA-seq datasets 6,8,42 . From 4 SIS of mouse ST and PFC, we identified only 5 Shank3 transcripts (ranging 5,625-6,463 bp) in ST, with none detected in PFC upon validation.…”

“…Disruption in transcript-specific regulatory elements due to DNA mutations can lead to diseases. Transcriptome-wide changes are implicated in neuropsychiatric conditions, including autism spectrum disorder (ASD) [5][6][7][8] .…”

An atlas of expressed transcripts in the prenatal and postnatal human cortex

“…Alternative splicing (AS) is a post-transcriptional regulatory mechanism that acts to generate distinct mRNA molecules from a single pre-mRNA molecule. It plays an important role in development of the vertebrate central nervous system 1 with altered RNA splicing being implicated in numerous neurodevelopmental disorders 2,3 . Long-read transcript sequencing has facilitated the unambiguous characterization of full-length isoforms 4,5 , enabling the identification of novel transcript structures and coding regions that potentially harbour genetic variants predisposing to disease.…”