Two structurally similar NF-κB-inhibitor-interacting Ras-like proteins (NKIRAS) regulate the activity of the transcription factor NF-κB and thereby control several early immune mechanisms in mammals. We identified the orthologous sequences of NKIRAS1 and NKIRAS2 from the rainbow trout Oncorhynchus mykiss. The level of sequence identity was similarly high (≥68%) between the two and in comparison to their mammalian counterparts. Strikingly, NKIRAS2 was present as four transcript variants. These variants differed only in length and in the nucleotide composition of their 5’ termini and were most likely generated by splicing along unconventional splice sites. The shortest NKIRAS2 variant was most strongly expressed in a lymphocyte-enriched population, while NKIRAS1 was most strongly expressed in cells of myeloid origin. Fluorescent-labelled NKIRAS1 and NKIRAS2 proteins from rainbow trout were detected in close association with the p65 subunit of NF-κB in the nucleus and cytoplasm of CHSE-214 cells. Subsequent reporter-gene experiments revealed that NKIRAS1 and a longer NKIRAS2 variant in rainbow trout decreased the level of activated NF-κB, while the two shortest NKIRAS2 variants increased the NF-κB activity. In addition, the overexpression of the shortest NKIRAS2 variant in CHSE-214 cells induced a stronger transcription of the genes encoding the pro-inflammatory cytokines TNF, CXCL8, and IL1B compared to non-transfected control cells. This is the first characterisation of NKIRAS orthologues in bony fish and provides additional information to the as yet underexplored inhibition pathways of NF-κB in lower vertebrates.

两种结构相似的与NF-κB抑制剂相互作用的Ras样蛋白（NKIRAS）调节转录因子NF-κB的活性，从而控制哺乳动物的几种早期免疫机制。我们从虹鳟Onkihynchus mykiss中鉴定了NKIRAS1和NKIRAS2的同源序列。两者之间的序列同一性水平较高（≥68％），与之相比，它们的哺乳动物相同。令人惊讶的是，NKIRAS2作为四个转录物变体存在。这些变体仅在长度和5'末端的核苷酸组成上有所不同，并且很可能是通过沿非常规剪接位点进行剪接而产生的。最短的NKIRAS2变异体在淋巴细胞丰富的人群中表达最强，而NKIRAS1在髓样来源的细胞中最强烈表达。检测到虹鳟鱼的荧光标记NKIRAS1和NKIRAS2蛋白与CHSE-214细胞核和细胞质中NF-κB的p65亚基紧密相关。随后的记者基因实验表明，虹鳟鱼中的NKIRAS1和更长的NKIRAS2变体降低了活化的NF-κB的水平，而两个最短的NKIRAS2变体则提高了NF-κB的活性。此外，与未转染的对照细胞相比，CHSE-214细胞中最短的NKIRAS2变体的过表达诱导了编码促炎细胞因子TNF，CXCL8和IL1B的基因的转录更强。