Alternative splicing of key signal transduction molecules drives monocyte-to-macrophage differentiation.

Lesole, P., Meyer, V., Gentle, N.*

School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa

Monocytes and macrophages are important cells in the innate immune system, defending against pathogen entry. A key property of monocytes is their ability to infiltrate tissue injury sites and differentiate into macrophages, a process associated with transcriptomic changes. However the involvement of post-transcriptional modifications such as alternative splicing (AS) in this process have not yet been fully characterised. AS analysis is categorised into isoform- and count-based methods, where the latter infers mRNA isoforms using disjointed counting bins obtained from isoform-specific reads, while differential exon (DEU) and transcript (DTU) usage and differential alternative splicing (DAS) event analysis are key count-based methods essential for identifying specific AS events. In vitro studies of monocyte-to-macrophage differentiation utilise promonocytic cell lines such as THP-1, stimulated with phorbol 12-myristate-13-acetate (PMA) to overcome the challenges of culturing primary monocytes. As this study aimed to identify DEU, DTU and DAS events during monocyte-to-macrophage differentiation and explore their interactions and contributions to transcriptional changes, we analysed RNA-seq data from THP-1 cells treated with PMA for 96 hours and untreated controls using DEXSeq for DEU (p-adj value < 0.05 and exon log2FC > 1), DRIMSeq for DTU (overall FDR < 0.05 at gene- and transcript-level using stageR) and rMATS-turbo for DAS (FDR < 0.05 and ΔPSI > 0.1) analysis. We identify 451 DEU, 679 DTU, and 238 DAS events, involving 160, 211 and 196 genes respectively. A number of these AS events were found to occur in genes encoding proteins with GTPase activity, highlighting the importance of these enzymes and their associated signalling pathways in cellular differentiation.

Keywords: alternative splicing, innate immunity, cellular differentiation, differential exon usage, differential transcript usage, differential splicing analysis, RNA-seq analysis