Liu Chen, Lou Chih-Hong, Shah Vrutant, Ritter Ruth, Talley Julia, Soibam Benjamin, Benham Ashley, Zhu Huifeng, Perez Eloy, Shieh Yi-Er, Gunaratne Preethi H, Sater Amy K
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
Department of Stem Cell Engineering, Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77225, United States.
Dev Biol. 2016 Jan 1;409(1):26-38. doi: 10.1016/j.ydbio.2015.11.005. Epub 2015 Nov 6.
MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.
已知微小RNA（miRNA）在脊椎动物发育调控中发挥多种作用。为了研究胚胎发育过程中miRNA与靶mRNA的关系，我们进行了小RNA测序，以鉴定非洲爪蟾早期原肠胚中表达的miRNA。我们共鉴定出180种miRNA，并确定了这些miRNA前体序列在非洲爪蟾基因组中的位置。在这些miRNA中，有141种是先前在热带爪蟾中鉴定出的miR。与人类miRNA比对后，我们鉴定出39种此前未在爪蟾中描述过的miRNA。我们还采用生化方法分离了早期原肠胚中与RNA诱导沉默复合体（RISC）相关的mRNA，这些mRNA因此成为miRNA依赖性调控的候选靶标。通过逆转录聚合酶链反应（RT-PCR）对这个与RISC相关的mRNA库进行分析表明，许多对早期模式形成和细胞分化至关重要的基因可能受到miRNA的调控。Smad1转录本与RISC相关；靶标预测算法在Smad1a和Smad1b的3'非翻译区（UTR）中识别出一个共同的、针对miR-26 的单一miRNA结合位点。通过靶向保护吗啉代寡核苷酸（TPMO）破坏miR-26与Smad1 3'UTR之间的相互作用，会导致Smad1蛋白积累增加2倍，BMP4/Smad1靶基因的表达适度增加，组织者基因的表达减少，并且在约25%的胚胎中出现部分腹侧化表型。miR-26的过表达导致Smad1依赖性基因的表达适度降低，以及表达组织者基因not1的区域扩大。我们的研究结果表明，miR-26与Smad1 3'UTR之间的相互作用在轴向模式形成过程中调节Smad1的功能；它们也为原肠胚形成过程中miRNA及其调控相互作用的功能分析奠定了基础。
