Single-cell transcriptional footprint for pseudogene SsCLEC9A is associated with antigen processing and presentation in Sus scrofa.

Abstract

The C-type lectin domain family 9 member A (CLEC9A) is widely recognized as the most critical receptor protein for cross presentation of dead cell associated antigens in animal dendritic cells (DCs). Surprisingly, we revealed for the first time that the sole CLEC9A (SsCLEC9A) in pigs becomes a pseudogene due to three causal mutations that occurred approximately 29.8-44.7 million years ago, challenging the significance of CLEC9A in immune cross-presentation across mammals. Interestingly, we found that SsCLEC9A can transcribe a mutated transcript encoding a truncated protein. Through fluorescence-activated cell sorting and single-cell RNA sequencing, we observed that SsCLEC9A mutant transcript is mainly expressed in DCs and correlated with the expression of its homolog CLEC7A. Further data showed that DCs with SsCLEC9A mutant transcripts exhibited reduced cellular interaction ability and downregulation of antigen presentation function, displaying the characteristics of mature DCs. In addition, introducing the conserved sequence of CLEC9A gene into FLT3L-induced bone marrow hematopoietic cells significantly increased the expression of genes involved in antigen processing and presentation. This study presents a natural mutation model of pseudogenes to understand its transcriptional adation, and provides a fundamental basis for rescuing SsCLEC9A to promote immunity in pigs in the future.