Long-read transcriptome profiling of germinal centre B cells
Alternative splicing (AS) is a major regulatory process underpinning the development and function of the immune system. 29% of alternatively spliced genes are specific to immune cells, yet the regulation and function of AS during B cell activation remain largely unknown. The activation of naive B cells and the subsequent germinal centre (GC) reaction drive the cascade of reactions resulting in short- and long-term antibody responses, making the GC crucial for adaptive immunity. However, abnormal function of GC B cells contributes to autoimmune disease and the development of lymphomas. Hence, GC reaction needs to be tightly regulated.
Previous studies have linked individual AS events in GC B cells to B cell malignancies using short-read sequencing; however, this methodology is limited in defining the complete sequence of transcript variants generated by AS. Therefore, many transcript variants remain undefined. During my PhD, I have developed a long-read sequencing methodology Oxford Nanopore Technologies (ONT) workflow to understand post-transcriptional regulation at both gene and isoform levels in human and mouse GC B cells. Because one of the challenges of ONT is the accurate computational analysis of isoforms, we developed the ‘Nexons’ pipeline to identify the differentially spliced transcript variants.
Transcriptome characterisation using ONT allowed us to detect differentially expressed transcripts during antigen-mediated activation of GC B cells in human and mouse. Moreover, identification of individual isoforms with Nexons revealed differential splicing of transcripts, including potentially novel splice variants, as well as changes which were undetectable in short-read sequencing data. An in-depth analysis revealed the differential regulation of poison exons (PE) in serine/arginine-rich splicing factors (SRSF) (e.g., SRSF3 and SRSF7). Naive B cells preferentially expressed isoforms carrying PE, leading to nonsense-mediated mRNA decay, whilst the PE were preferentially removed in activated and GC B cells. Notably, we found this regulation of PE in splicing factors is conserved between human and mouse. We validate an ONT/Nexons workflow as a suitable method for the identification and quantification of transcript isoforms and highlight the SRSF family as important candidates for regulating the GC reaction.