Identification of splice isoforms in T helper cells using Nanopore-based cDNA sequencing

Abstract

Abstract The T helper (Th) cell compartment of the adaptive immune response provides a central mechanism of balance between inflammation and immune tolerance. The differentiation of naïve Th cells into both regulatory and effector Th subsets is regulated through complex gene regulatory networks that lead to distinct transcriptomes. Alternative RNA splicing is a major component of cellular differentiation that allows protein heterogeneity and mechanistic diversity across cell types. However, our understanding of alternative splicing control is still limited. Here we show differential expression of RNA binding Proteins (RBP) genes among Th cell subsets suggesting a regulatory role of RBPs in RNA splicing and Th differentiation. In order to interrogate the regulatory gene network, we utilized innovative Nanopore-based sequencing to generate a single molecule transcriptome map of naïve CD4 T cells, and differentiated Treg and Th1 cells to characterize full-length cDNA transcripts and alternatively spliced isoforms. Gene expression was compared across four major gene categories; RBPs, Transcription Factors (TFs), Other Protein Coding (OPC) and LncRNA. Our study revealed that RBPs exhibited high splicing potential while LncRNA exhibited relatively less splicing potential in comparison to other gene categories in all cell types. Overall, we identify RBPs that are both differentially expressed in Th subsets and differentially spliced themselves. This might represent an important control mechanism of functional protein diversity in regulating immune responses.