Comparative analyses of analyses of splicing and isoform variants by short and long-read sequencing

Resumen

FUS mutations cause aggressive, early-onset Amyotrophic lateral sclerosis (ALS) and a form of dementia frontotemporal (FTD) disorders. Although the effect of FUS mutations on motor neuron degeneration has been largely investigated, the more global systemic consequences of these mutations, particularly on the RNA-processing landscape in a variety of other tissues, are not fully characterized. In the present work, we investigated transcriptomic alterations driven by the homozygous FUSDelta14 mutation using a knock-in mouse model.

We re-analysed the transcriptomic data from the RNA sequencing (RNA-Seq) by short read (Illumina) in four tissues (brain, spinal cord, liver, and skeletal muscle) of the control and mutant mice and develop the best pipeline for gene expression (different references) and splicing analysis (rMATs Turbo and IsoformSwitchAnalyzer). Further, to overcome inherent short-read limitations, we optimised the sequencing of cDNA using the long read technology from Oxford Nanopore, from library preparation through data analysis, enabling direct comparison between sequencing platforms.

Most changes in gene expression were tissue-dependent, with shared themes around RNA metabolism, lipid transport, mitochondrial activity and glial development. Fus and several pseudogenes of ribosomal genes were repeatedly dysregulated across all tissues. Splicing analysis via rMATS uncovered hundreds of genotype-specific events, primarily exon skipping and intron retention, including the skipping of the exon 14, what characterise the mouse model. However, the transcript-level quantification highlighted discrepancies in this reconstruction. As a first approximation, nanopore-based long-read RNA sequencing of the hippocampus seems to allow us to identify full-length isoforms and novel transcripts, demonstrating its effectiveness in dissecting complex splicing landscapes.

In summary, the present study reveals that the FUSDelta14 mutation causes widespread changes in gene expression and RNA splicing in a tissue-restricted manner. These studies indicate that long-read sequencing includes an essential source of resolution in identifying functionally relevant isoform changes and acts as a potent method to dissect transcriptomic dysregulation in ALS-FTD models.