Expanding the avian micro RNA repertoire of domesticated poultry and investigating the NLRP3 inflammasome messenger RNA and micro RNA transcriptomes in Anas platyrhynchos (ducks)

Abstract
Micro RNAs (miRNAs) are a class of short (~22 nucleotides) non-coding RNAs that function as post-transcriptional gene silencers. Currently only chickens and zebra finch contribute to the known repertoire of avian miRNAs and it is likely that large gaps exist in the knowledgebase required to explore miRNA regulation of avian biological processes. With the availability of the turkey and duck genomes, we chose to define the miRNA profile from embryonic turkey brain and bursa of Fabricius tissues as well as embryonic duck intestine and spleen tissues to expand on the avian miRNA repertoire. We identified 436 and 329 unique miRNAs from miRNA-SEQ libraries prepared from embryonic turkey tissues and embryonic duck tissues respectively. Sequencing results were validated with qRT-PCR. Using datamining, critical miRNA profiles were characterized for tissues by performing literature review on miRNAs that were either substantially abundant (top 10 abundant miRNAs from a given tissue), differentially expressed > 2-fold, or identified as a tissue-associated miRNA through text mining. ☐ Additionally, the involvement of miRNAs in inflammation is still a developing area of interest. Inflammation is the normal response of a tissue to infection or tissue damage. To neutralize the causative agent and restore homeostasis, the innate immune system initiates a multiphase program initiated by inflammasomes. The NLRP3 inflammasome is the most studied mammalian inflammasome. However, no studies on the NLRP3 inflammasome have been performed in avians, including ducks. Furthermore, miRNA regulation of NLRP3 inflammation is largely unexplored. We chose to characterize miRNA and RNA gene expression in duck primary monocytes exposed to nigericin, a bacterial toxin derived from Streptomyces hygroscopicus and NLRP3 agonist, by performing miRNA-SEQ and RNA-SEQ. We identified 514 unique miRNAs from miRNA-SEQ libraries prepared from duck primary monocytes 1, 2, and 4 hours after exposure to nigericin including an untreated control. Sequencing was validated with qRT-PCR. Using datamining, critical miRNA profiles were characterized for samples by performing literature review on miRNAs that were either substantially abundant (top 10 abundant miRNAs), differentially expressed > 2-fold compared to the untreated control, or identified as a monocyte-associated miRNA through text mining. Increasing the miRNA knowledge base in poultry paves the way for future studies of poultry biological processes and may lead to discoveries in poultry health and development. Furthermore, by characterizing miRNAs in duck primary monocytes, a cell type that expresses NLRP3, and studying the regulation of NLRP3 inflammation, this investigation will help define the role of miRNAs in a pro-inflammatory state.
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Keywords
Biological sciences, Avian, Inflammasome, Inflammation, Mirnas, NLRP3, Sequencing
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