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CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation
Qinghua Zeng1, Jingjing Cao2, Fei Xie3
1Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Zhimin Street, Qingshan Lake, Nanchang, 330045, P.R. China.
Abstract:
Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-β activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5-/- DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5-/- DF-1 cells had increased IFN-β production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-β production and interferon-stimulated gene expression.
Insights
Chicken PRMT5 protein suppresses interferon-beta (IFN-β) activation. Knocking out the PRMT5 gene in chicken cells increased IFN-β production, revealing its role in immune suppression.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Protein arginine methyltransferase 5 (PRMT5) is a key enzyme in arginine dimethylation, with known functions in mammalian cells.
- The specific role and regulation of PRMT5 in chicken cellular immunity, particularly concerning interferon pathways, remain largely unexplored.
Purpose of the Study:
- To investigate the expression, localization, and function of chicken PRMT5 (chPRMT5) in chicken cells.
- To elucidate the role of chPRMT5 in the regulation of the chicken interferon-beta (chIFN-β) pathway and innate immune responses.
Main Methods:
- Western blotting and immunofluorescence to determine chPRMT5 expression and localization in various chicken tissues.
- CRISPR/Cas9 gene editing to create a PRMT5-deficient DF-1 cell line (prmt5-/-).
- Luciferase reporter assays and overexpression studies to assess chIFN-β activation and interferon-stimulated gene expression.
Main Results:
- Chicken PRMT5 is expressed across multiple tissues and localizes to both cytoplasm and nucleus.
- Ectopic expression of chPRMT5 inhibited chMDA5-induced chIFN-β activation.
- PRMT5-deficient DF-1 cells exhibited normal morphology and proliferation but showed significantly increased IFN-β production compared to wild-type cells.
- chPRMT5 was found to suppress IFN-β production and interferon-stimulated gene expression.
Conclusions:
- Chicken PRMT5 plays a significant role in suppressing the innate immune response by inhibiting IFN-β production.
- This study establishes a functional link between chPRMT5 and the regulation of interferon pathways in chicken cells, providing a foundation for further research into avian immunology.
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