At Least Two Genes Encode Many Variants of Irak3 in Rainbow Trout, but Neither the Full-Length Factor Nor Its Variants Interfere Directly With the TLR-Mediated Stimulation of Inflammation

Abstract

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout (Oncorhynchus mykiss), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3-sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms.

The expression of myogenic gene markers during the embryo‐larval‐transition in Pikeperch ( Sander lucioperca )

“…Transcriptional analysis was conducted using a quantitative real‐time PCR based on a microfluid circuit system (Biomark, FLUIDIGM) using EvaGreen fluorescence dyes (Bio‐Rad). The protocol was described in detail by Rebl et al (2019). The design of Fluidigm 48.48 Dynamic Array IFC allows the parallel quantification of 48 transcripts in 48 samples each.…”

PIAS Factors from Rainbow Trout Control NF-κB- and STAT-Dependent Gene Expression

“…In previous studies, we inspected the diversity of ohnologue and paralogue genes in salmonid fish that encode immune inhibitors [ 14 ]. We repeatedly found that structural modifications of the protein may fundamentally impact its function [ 46 , 47 ]. However, all three Pias1 proteins from rainbow trout strongly resemble the prototypical PIAS architecture of their human orthologue.…”

Riding the wave of innovation: immunoinformatics in fish disease control

“…This complex then phosphorylates IRAK2 or IRAK1 and recruits TRAF6 (tumour necrosis factor receptor-associated factor 6) via ubiquitination ( Cao, Henzel & Gao, 1996 ; Fitzgerald & Kagan, 2020 ). Studies have reported that IRAK2 has been lost or not identified in fish ( Zhang et al, 2014 ; Rebl et al, 2019 ), however, another study has stated that IRAK2 was found in the West Indian Ocean coelacanth ( Latimeria chalumnae ) genome ( Li et al, 2018 ). Following ubiquitination, TRAF6 interacts and activates the TAB1/TAK1/TAB2 complex whereby TAB1 activates TAK1 (transforming growth factor- β -activated kinase 1) while TAB2 serves as an adaptor that connects TAK1 to TRAF6.…”