Genetic and functional analysis of unproductive splicing using LeafCutter2.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Alternative splicing generates unproductive mRNA transcripts with premature termination codons, leading to degradation via nonsense-mediated decay (NMD).
• These events can decrease protein expression, influencing gene regulation and disease pathogenesis.
• Current methods for analyzing unproductive splicing from RNA sequencing data are limited.

Purpose of the Study:

• To introduce LeafCutter2, a novel computational tool for identifying and quantifying unproductive splicing events from short-read RNA sequencing data.
• To investigate the landscape and regulation of unproductive splicing across species, tissues, and developmental stages.
• To explore the role of unproductive splicing in Alzheimer's disease (AD) and its genetic underpinnings.

Main Methods:

• LeafCutter2 utilizes minimal gene annotations (start and stop codons) to identify NMD-inducing splicing events.
• The tool enables differential analysis of unproductive splicing between sample groups and maps unproductive splicing quantitative trait loci (u-sQTLs).
• Application to RNA-seq data from multiple human and non-human species, including AD patient cohorts.

Main Results:

• LeafCutter2 successfully identified and quantified unproductive splicing events across diverse species and tissues, revealing significant variation.
• A conserved, developmentally regulated increase in unproductive splicing was observed during testis maturation.
• Unproductive splicing events were identified in 18 AD risk genes, suggesting a role in mediating genetic risk for Alzheimer's disease.

Conclusions:

• LeafCutter2 is a powerful tool for analyzing unproductive splicing and its functional consequences on gene expression.
• Unproductive splicing plays a significant role in gene regulation, development, and disease, offering insights beyond traditional expression quantitative trait loci (eQTL) analyses.
• The findings highlight the importance of considering unproductive splicing in understanding genetic contributions to complex diseases like AD.