Going through changes: spatial technology reveals pubescent RNA regulation in the brain
A new spatial transcriptomic approach captures the regulation of splicing and polyadenylation sites during pubescent brain development at near-single-cell resolution.
An international collaboration, led by researchers from Weill Cornell University (NY, USA), has developed a new spatial approach to investigate critical RNA processing steps in the brain, both before and after puberty. This approach, combining long- and short-read sequencing with an innovative spatial technology, overcomes the challenges of studying these processes within their biological context, offering greater insight into brain development.
Puberty represents a period of developmental change, which can affect brain plasticity characterized by changes in RNA splicing and polyadenylation. However, investigating which areas of the brain are most strongly altered by these modifications has been a challenge, due to a reliance on low-resolution technologies with insufficient transcript capture efficiency.
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To overcome the limitations of traditional methods, the researchers have combined long- and short-read sequencing with Slide-SeqV2 (Seeker™ technology) to observe the developmental regulation of splicing and polyadenylation that occurs in specific cortical layers and cell types during puberty. Their running of both long- and short-read sequencing in tandem meant that they were able to describe transcript information spanning multiple exons while also conducting gene-expression based analyses, offering the greatest transcriptomic insight into the tissue.
Their cell type-specific spatial transcriptomic approach is called spatial isoform sequencing (Spl-ISO-Seq); it spatially barcodes RNA molecules based on tissue placement, allowing for precise isoform quantification and near-single-cell resolution that together provide a clearer picture of transcript diversity within tissues. Spl-ISO-Seq achieves this by enriching for long, exon-spanning cDNAs, which results in much longer read lengths than standard preparations. They also developed a complementary software suite that enables barcode deconvolution and isoform expression analysis to assist with this process.
Applying their approach to prepubescent and postpubescent post-mortem visual cortex tissue samples, they found developmental differences in RNA regulation in different areas of the brain. Splicing and polyadenylation regulation was stronger in the cortex than in white matter, with cortical layer four showing the most developmentally regulated splicing changes in excitatory neurons and poly(A) sites among the cortical layers. Downstream of this regulation, the researchers noted an enrichment with repetitive elements, which have been shown to be expressed throughout the brain during development. However, they observed that oligodendrocyte regulation was stronger in white matter than in cortical layers. Furthermore, they recognized an association between splicing changes and postsynaptic structure and function.
This study provides an interesting case for deploying spatial transcriptomic technology to investigate gene regulation in development. It also demonstrates the advantages of using this technology for mapping how RNA processing shapes tissue function, with applications beyond development, such as neurodegeneration and cancer.