Splicing-aware scRNA-Seq resolution reveals execution-ready programs in effector Tregs.

Area of Science:

• Molecular Biology
• Genomics
• Immunology

Background:

• Single-cell RNA sequencing (scRNA-Seq) is crucial for cell biology research.
• Current scRNA-Seq methods lack the ability to differentiate between spliced and unspliced mRNA during dimensionality reduction.
• Unspliced mRNA indicates transitional cell states, vital for understanding dynamic biological processes like regeneration.

Purpose of the Study:

• To develop a novel method, SANSARA (Splicing-Aware scrNa-Seq AppRoAch), for analyzing scRNA-Seq data with splicing awareness.
• To enable the distinction between spliced and unspliced mRNA forms in scRNA-Seq analysis.
• To uncover new dimensions for discovery in scRNA-Seq-based research.

Main Methods:

• Development of the SANSARA method for splicing-aware analysis of scRNA-Seq data.
• Application of SANSARA to characterize peripheral blood regulatory T cell (Treg) subsets.
• Analysis of splicing heterogeneity in Th1 and cytotoxic CD4+ T cell subsets.

Main Results:

• SANSARA revealed a complementary interplay between FOXP3 and Helios in Tregs.
• High levels of spliced IL10RA, LGALS3, FCRL3, CD38, ITGAL, and LEF1 mRNAs were identified in effector Tregs.
• Substantial splicing heterogeneity was observed across subset-specific genes in Th1 and cytotoxic CD4+ T cells.

Conclusions:

• SANSARA provides a straightforward implementation for current scRNA-Seq pipelines.
• The method opens new avenues for discoveries by incorporating splicing information.
• Functional cell subsets may differ in actively produced and stored mRNA forms, impacting cellular function.