Functional assessment of 3'UTR regulatory elements using the zebrafish (Danio rerio) model system - towards modulating protein levels in Atlantic salmon (Salmo salar) for more sustainable aquaculture

• Main Author: Barvik, Morten
• Format: Master Thesis
• Language: English
• Published: The University of Bergen 2021
• Subjects: zebrafish; 3'UTR; mRNA stability; zebrafish injection; salmon; 759929; Atlantic salmon; Salmo salar
• Online Access: https://hdl.handle.net/11250/2834593

Post-transcriptional regulation is a complex process that encompasses a myriad of different structures and mechanisms. There are several indications that regulatory elements in the 3'UTR play an important role in the stability of mRNA transcripts. Sequences of 60 nucleotide base pairs from the 3'UTR of two genes, trip10a from zebrafish and mx2.2 from Atlantic salmon, were identified to contain Adenylate-uridylate-rich elements (AU-rich elements; AREs). AREs are proposed to have a destabilizing effect on mRNA stability. We mutated the identified AREs, and inserted both unmutated wild type (WT) and mutated 60 bp sequence elements into the 3'UTR of a GFP expression construct. Zebrafish embryos were subjected to two different injection experiments. In the first experiment embryos were injected with GFP mRNA containing WT and mutated 3'UTR elements from the trip10a gene. In the second experiment embryos were injected with GFP mRNA containing WT and two different mutated versions of 3'UTR elements from the mx2.2 gene. Zebrafish embryos were injected at the 1-cell stage and measured for fluorescence intensity at 6, 12, 24, 48, 72, 96, and 120 hpf. GFP Fluorescence was normalized by injecting embryos with 75 ng/μL mCherry RFP mRNA and mRNA stability was assessed by detection of GFP and RFP fluorescence signals. The mutated trip10a 3'UTR caused a significant increase in GFP levels (p < 0.01). Mutations in two AREs (Mutation A) from the mx2.2 3'UTR caused a significant increase in GFP levels (p < 0.05). Mutations in two AREs and a selection of AT repeats (Mutation B) caused a significant increase in GFP levels (p < 0.05). These results support the approach in this thesis as a robust method for using zebrafish as a model to assess mRNA stability. This method can be used to test and assess putative regulatory elements for mRNA stability in salmon, and subsequently used to modulate expression of key anti-viral proteins to make salmon more robust against disease. This can thus be a small step towards a more ...