Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis
Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis
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DOI：
10.1016/j.bbrc.2009.02.122
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发表时间：
2009-04-17
影响因子：
3.1
通讯作者：
Liu, Ting Xi
中科院分区：
文献类型：
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作者：
Du, Ting-Ting;Fu, Yan-Fang;Liu, Ting Xi
微小RNA（microRNAs，miRNAs）是一类内源性非蛋白质编码小分子RNA，通过与靶转录本结合进行切割或翻译抑制，在动植物体内发挥重要的调控作用。尽管越来越多的基因被预测为miRNA靶基因，通过生物信息学分析和体外报告基因分析（RA-In Vitro），但其中很少有在生理背景下被实验验证的。利用体内瞬时报告基因分析（TRA-In Vivo），我们分别鉴定了羟甲基胆烷合酶B（hmbsb）和Kruppel样转录因子d（klfd）为miR-451和miR-144的潜在靶基因。尽管hmbsb、miR-451、klfd和miR-144在斑马鱼红细胞生成过程中在发育中的红系祖细胞中均共表达，但使用基于转基因的体内生理报告基因测定（PRA-In Vivo），仅klfd可被验证为miR-144的真正生理靶标。因此，未能在生理学背景下验证hmbsb作为miR-451靶标提出了关于如何确定miRNA的真正靶标的关键问题。这些结果强调了使用多种方法结合Western印迹分析来验证给定miRNA的生理靶点的重要性。(C)2009 Elsevier Inc. All rights reserved.
MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. Despite increasing number of genes has been predicted to be miRNA targets by bioinformatics analysis and luciferase-based reporter assay in vitro (RA-In Vitro), few of them have been experimentally validated in physiological context. Using transient reporter assay in vivo (TRA-In Vivo), we identified hydroxymethylbilane synthase b (hmbsb) and Kruppel-like transcription factor d (klfd) as potential target gene for miR-451 and miR-144, respectively. Although hmbsb, miR-451, klfd and miR-144 are all co-expressed in the developing erythroid progenitors during zebrafish erythropoiesis, only klfd can be validated as a bona fide physiological target of miR-144 using a transgene-based physiological reporter assay in vivo (PRA-In Vivo). Thus, failure to verify hmbsb as miR-451 target in physiological context raises a crucial question as to how to determine bona fide target of miRNA. The results address the importance of using multiple approaches combined with Western blot analysis to validate the physiological target of a given miRNA. (C) 2009 Elsevier Inc. All rights reserved.