Reverse engineering neuron-type-specific and type-orthogonal splicing-regulatory networks using diverse cellular...

Area of Science:

• Neuroscience
• Molecular Biology
• Computational Biology

Background:

• Alternative splicing (AS) generates diverse gene isoforms crucial for neuron type-specific functions.
• Existing research on RNA-binding proteins (RBPs) and AS is limited to a few neuron types, necessitating comprehensive analysis.

Purpose of the Study:

• To create a holistic map of the alternative splicing regulatory landscape across 133 mouse neocortical cell types.
• To identify RNA-binding proteins (RBPs) and their cell-type-specific activities that control alternative splicing (AS).

Main Methods:

• Network reverse engineering was applied to pseudobulk transcriptomes from single-cell RNA sequencing data.
• Inferred regulons of 350 RBPs and their cell-type-specific activities.
• Validated Elavl2's role in medial ganglionic eminence (MGE)-specific splicing using an in vitro embryonic stem cell (ESC) differentiation system.

Main Results:

• A comprehensive map of the neuron-type-specific AS regulatory landscape was generated for 133 mouse neocortical cell types.
• The regulons and cell-type-specific activities of 350 RBPs were inferred.
• Elavl2 was validated as a key RBP for MGE-specific splicing in GABAergic interneurons.
• A module of exons and candidate regulators specific to long- and short-projection neurons was identified.

Conclusions:

• This study provides a valuable resource for understanding splicing regulatory programs that contribute to neuronal molecular diversity.
• The findings reveal splicing regulation that may not be captured by gene expression-based classifications.
• Identified RBPs and splicing modules offer insights into the mechanisms driving neuronal heterogeneity.