Natural Killer (NK) cell receptor expression in cattle: development of a pipeline for accurate quantification of RNA Sequencing (RNA-Seq) data

Q Science (General);QR180 Immunology

Borne, Richard ; Hammond, John

University:University of Glasgow

Department:School of Veterinary Medicine

关键词: Cattle, immunogenetics, LRC, NKC, RNA-Seq, KIR, KLR, NGS, bioinformatics, expression, transcriptomics, NK, natural killer.;   

Others  :  http://theses.gla.ac.uk/71955/7/2019bornephd.pdf

来源: University of Glasgow

【 摘 要 】

Cattle have undergone significant gene expansion within two of the major NK receptor encoding complexes, the leukocyte receptor complex (LRC) and natural killer complex (NKC). This expansion has resulted in a number of highly similar genes densely packed within each complex. The genes can be highly polymorphic and encode for fundamentally important receptors for controlling the functional response of NK cells. Understanding the nature of transcription of the LRC and NKC genes is required to confirm existing gene models, predicted functional status and also their distribution in cell and tissue types. RNA-Seq offers the potential for high-resolution analysis of LRC/NKC gene transcription. For this to occur, an analysis pipeline needed to be developed to account for the high sequence similarity and resulting multi-mapping of reads.In this project an analysis pipeline, UniMMap, has been created that utilises the concept of mappability to weight the contribution of multi-mapping reads to the total read count. UniMMap was utilised to assess transcription of LRC/NKC genes in a number of RNA-Seq datasets. Transcription in peripheral blood mononuclear cells (PBMCs) and NK cells from two cattle was compared. Genes predicted to be non-functional based on the reference assembly were found to be consistently transcribed. Transcription was also compared between multiple immune cell types. CD8+ T cells were shown to transcribe killer-cell immunoglobulin-like receptor (KIR) at an equivalent level to NK cells. RNA-Seq data from multiple cattle tissues was analysed to produce an LRC/NKC gene atlas. Transcription of at least one LRC and NKC gene was observed in every tested tissue. Total transcription of the LRC/NKC genes was found to be highest in the lymph node, mammary gland and lung tissue. To assess the utility of UniMMap in a controlled study, as well as in a different species, the transcription of LRC/NKC genes were investigated in goats from a peste des petits ruminants virus (PPRV) vaccination study. A subset of the genes were found to respond differently in PBMCs between unvaccinated and vaccinated animals. This supports a role for these genes during PPRV infection.UniMMap has facilitated analysis of transcription of LRC/NKC genes from multiple species, cell/tissue types and RNA-Seq library types. The consistently observed transcription of predicted non-functional genes suggests the presence of functional alleles or possibly a role in regulating transcription of other genes within each complex. The changes observed in their transcription during infection, coupled with a similar distribution among tissues and immune cell types, suggests a functional similarity with their human counterparts.