Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish

Abstract

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.

Overview and New Insights Into the Diversity, Evolution, Role, and Regulation of Kisspeptins and Their Receptors in Teleost Fish

“…However, kisspeptin transcripts and proteins are widely distributed in various brain areas and tissues of fish, but no clear roles have been associated with these extra brain kisspeptins. These tissues/organs, include the pituitary gland, the spinal cord, the intestine, the gonads, and the liver ( 62 – 65 ). The presence of kisspeptin in blood has been detected in mammals ( 66 ).…”

“…It is important to mention that alternative splice variants are frequently observed in mammals when analyzing GPCR tissue distribution ( 67 ), and spliced variants of kisspeptin have also been detected in several teleosts. For example, the presence of alternative splice variants for kissr2 and/or kissr3 was observed in Senegalese sole ( 6 , 45 ), southern bluefin tuna, Thunnus maccoyii ( 68 ), yellowtail kingfish, Seriola lalandi ( 68 ), zebrafish ( 69 ), European eel ( 44 ) and pejerrey fish ( 65 ) through intron retention. Mechaly et al.…”

“…Mechaly et al. ( 6 , 45 , 65 ) indicate that truncated and likely non-functional proteins are produced in this manner. In two species, yellowtail kingfish ( 68 ) and zebrafish ( 69 ) alternative spliced variants were also generated by deletion of exons.…”

“…For a detailed description of the splicing events detected in kisspeptin receptors in fish see Mechaly et al. ( 45 , 65 ). In this regards, future studies are needed to evaluate whether truncated proteins are generated and whether this represents regulation by unproductive splicing, as has been described for arginine-serine-rich (SR) splicing factors in several organisms ( 70 ).…”

Hormonal and neuroendocrine control of reproductive function in teleost fish.

“…Furthermore, in goldfish, intraperitoneal injections of kiss1, but not kiss2, stimulated the release of LH in sexually mature females 102 . Also, it has been reported that kiss2 may have effects on food intake and growth function 88,96,115 . Furthermore, it can act as a link between food intake, energy homeostasis, and reproduction [116][117][118] .…”