Blencowe Lab Research Examines How Microexons Regulate Neurogenesis and Autism-Associated Gene Programs

MoGen Researchers Reveal How Microexons Contribute to Neurogenesis and Autism-Linked Pathways

Alternative splicing is a fundamental layer of gene regulation that allows a single gene to produce various forms of RNA and proteins. While advances in sequencing have made it possible to map these isoforms across tissues and disease states, understanding what individual splice variants actually do remains a major challenge.

A recent study published in Nature Communications this year, from the lab of Benjamin Blencowe at the University of Toronto addresses this gap by focusing on microexons, short RNA segments that are highly enriched in the nervous system and frequently dysregulated in Autism Spectrum Disorder. Led by PhD candidate Steven Dupas, the study introduces a new approach to examine how these elements function during brain development.

Why microexons matter

Microexons are very short exons (parts of genes that make it into the final messenger RNA transcribed from DNA), typically 3 to 27 nucleotides in length. Despite their size, they are highly conserved and play important roles in neuronal transcripts. Years of research from the Blencowe lab have shown that many microexons are dysregulated in individuals with Autism Spectrum Disorder, often due to disruption of the proteins that control splicing, such as SRRM4. However, the function of most microexons remains unclear because traditional gene perturbation approaches inactivate entire genes, whereas methods that specifically disrupt exons are only beginning to emerge.

CHyMErA-seq, a high-throughput method for screening microexon function

To address this problem, Dupas developed CHyMErA-seq, a screening platform that combines CRISPR-based genome editing with single-cell RNA sequencing. This system, which builds on previous work developed in the Blencowe and Moffat Labs, uses two nucleases, Cas9 and Cas12a, which act like molecular scissors to delete a specific exon along with its surrounding regulatory sequence, while leaving the rest of the gene intact. At the same time, each edited cell carries a molecular tag that is captured during sequencing, allowing researchers to link a given perturbation to its transcriptional effect in that cell. Using this approach, Dupas performed a pooled screen targeting dozens of neuronal microexons in mouse embryonic stem cells, which were then differentiated into neurons and gene expression changes were measured using single-cell RNA sequencing.

The screen showed that certain microexons act like brakes to ensure the correct timing of distinct states of neurogenesis, the process by which new neurons are formed in the brain; deletion of certain microexons essentially lead to the cells prematurely activating transcription of gene programs associated with neurogenesis. Intriguingly, the screen also revealed a microexon in a gene with the opposite function when its deletion led to the downregulation of neurogenesis genes, suggesting that microexons maybe have varied roles in regulating the timing of neuronal differentiation.

Links to autism-associated pathways

To further interpret these results, the team compared their data with gene expression datasets from human brain studies and found that transcriptional changes resulting from microexon deletion in certain genes were similar to groups of dysregulated genes previously implicated in Autism Spectrum Disorder, including pathways primarily involved in cytoskeletal organization and neuronal signaling. They also found that in early neural cells, deletion of certain microexons led to increased expression of autism-associated genes at stages where these genes should typically be suppressed.

Researcher spotlight: Steven Dupas

For Dupas, the study represents the culmination of his PhD work in the Blencowe lab; he led the project, which was performed in collaboration with members of the Blencowe and Moffat labs, including designing the screening platform, performing the experiments, and analyzing the data. The work also reflects the rapid development of single-cell perturbation approaches in recent years.

“When the project started, it was still a pretty nascent field,” he said. “There was a lot to learn about how to implement these kinds of screens in ways that can be scalable in future experiments, and about analysing this type of noisy data.”

Now in the final stages of his PhD, Dupas is preparing to defend his thesis later this year. He has recently completed an internship at Genentech, where he worked on related screening approaches that use imaging-based readouts to study cellular phenotypes.

“Taking a project from start to finish was a really valuable experience,” he said. “You learn how to think about both the science and the process behind it.”

Looking ahead: capturing isoform diversity

Beyond its immediate findings, the study points to a broader shift already underway in functional genomics. Large-scale perturbation screens are becoming more common, particularly those paired with single-cell sequencing, and being used to train models that aim to predict gene function across different biological contexts. However, most of this work still relies on removing entire genes, treating them as single units rather than collections of distinct isoforms. As Dupas noted, these methods are isoform-agnostic, and do not capture how different forms of the same exons could have various roles in disease contexts, and experiments using more comprehensive perturbation methods would help inform these models and their accuracies.

CHyMErA-seq offers a way around that limitation. By allowing researchers to perturb individual exons within a gene and read out the resultant transcriptional changes at single-cell resolution, the platform opens up a level of precision that has been difficult to achieve at scale.

This work was supported by the Simons Foundation and the Canadian Institutes of Health Research. Additional support was provided through graduate and postdoctoral fellowships from the Natural Sciences and Engineering Research Council of Canada and the C.H. Best Foundation. Benjamin Blencowe holds the Canada Research Chair in RNA Biology and Genomics and the University of Toronto Banbury Chair in Medical Research.