Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan.
Department of Biology and Chemistry, Texas A&M International University, 5201 University Blvd., Laredo, TX, 78041, USA.
Mar Biotechnol (NY). 2021 Oct;23(5):821-835. doi: 10.1007/s10126-021-10066-z. Epub 2021 Sep 6.
The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.
人类肌球蛋白重链基因 MYH14 包含一个内含子 microRNA，miR-499。我们之前的研究表明，硬骨鱼类的 myh14/miR-499 具有不同的基因组组织和表达模式；然而，其调控机制尚不完全清楚。在本研究中，我们报告了斑马鱼 Danio rerio 中 myh14 表达的调控。斑马鱼 myh14 有三个同源基因，myh14-1、myh14-2 和 myh14-3。详细的启动子分析表明，myh14-1 的 5' 侧翼区 5710bp 和 myh14-3 的 5641bp 区域包含一个必要的调控区，可在胚胎发育过程中重新表达特定的表达模式。斑马鱼 myh14-1 的 5' 侧翼区及其 torafugu 同源物共享两个远端和一个近端保守区。这两个远端保守区对斑马鱼 myh14-1 的表达没有影响，但对 torafugu 的表达有影响，这表明 myh14 同源物之间存在替代的调控机制。myh14 同源物的 5' 侧翼区比较表明存在两个保守区。缺失这些保守区显著降低了 myh14-3 的启动子活性，但对 myh14-1 没有影响，表明 myh14 同源物具有不同的顺式调控机制。miR-499 的功能丧失导致胚胎发育中慢肌纤维的显著减少。本研究鉴定了不同的顺式调控机制来控制 myh14/miR-499 的表达，以及 miR-499 在斑马鱼肌肉纤维类型特化中的不可或缺作用。
