AUTHOR=Zhu Ying , Shi Rui , Yang Qian , Zhang Mengqian , Chen Songlin , Wang Na 

TITLE=Two Nipped-B-Like Protein A (Nipbla) Gametologs in Chinese Tongue Sole (Cynoglossus semilaevis): The Identification of Alternative Splicing, Expression Pattern, and Promoter Activity Analysis

JOURNAL=Frontiers in Marine Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/marine-science/articles/10.3389/fmars.2022.833070

DOI=10.3389/fmars.2022.833070

ISSN=2296-7745

ABSTRACT=<p>In mammals, the mutation of nipped-B-like protein (nipbl) leads to Cornelia de Lange Syndrome (CdLS), characterized by low birth weight, short stature, and structural abnormalities of the skeleton, heart, and gut. In Chinese tongue sole (<italic>Cynoglossus semilaevis</italic>), a typical marine fish exhibiting sexual size dimorphism, the <italic>nipbl</italic> homolog gene (nipped-B-like protein A (nipbla)) was also screened with female higher expression level by somatotropic and reproductive tissues’ transcriptomic analysis. In this study, two nipbla genes, namely, <italic>nipbla-w</italic> and <italic>nipbla-z</italic>, were identified from the W and Z chromosomes of <italic>C. semilaevis</italic>, respectively. Similar to other mammalian and fish species nipbl, the two homolog proteins of <italic>C. semilaevis</italic> contained two conserved domains, namely, cohesion_HEAT and Nipped-B_C. The phylogenetic tree analysis showed that these two nipbla gametolog proteins were first clustered together and then grouped with other fish species. At least two types of alternative splicing sites were observed in exon 12 of the <italic>nipbla-z</italic> gene, which produced <italic>nipbla-z-tv1</italic> and <italic>nipbla-z-tv2</italic>. Also, the sex-biased expression patterns of different <italic>nipbla-w</italic> and <italic>nipbla-z</italic> transcripts in female and male tissues were revealed by quantitative PCR (qPCR). The highest expression level of <italic>nipbla-w</italic> was observed in female gonad. While <italic>nipbla-z-tv1</italic> exhibited relatively high expression in the muscle, liver, gonad, and brain, <italic>nipbla-z-tv2</italic> only showed its expression superiority in the muscle of male individuals. The promoter regions of <italic>nipbla</italic> genes were amplified, and their transcription activity was successfully verified by a dual-luciferase reporter system. After <italic>nipbla-w</italic> and <italic>nipbla-z</italic> knockdown in the brain cell lines by RNA interference, a series of growth-related genes were influenced, including Bone Morphogenetic Protein 4 (<italic>bmp4</italic>), Wnt Family Member 11 (<italic>wnt11</italic>), and Sprouty Related EVH1 Domain Containing 2 (<italic>spred2</italic>). The prediction of transcription factors suggested that c-Jun, sex-determining region Y (SRY), POU Class 1 Homeobox 1 (POU1F1a), myogenic differentiation antigen (MyoD), signal transducer and activator of transcription 5a (STAT5A), and nuclear factor I C (Nfic) might be the putative upstream regulatory factors for <italic>nipbla</italic>; among them, c-Jun has been verified to effectively regulate the transcriptional activity of <italic>nipbla</italic>. The identification of two <italic>nipbla</italic> genes provided important data for interpreting the sexual size dimorphism in <italic>C. semilaevis.</italic></p>