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  • Coherent but overlapping expression of microRNAs and their targets during vertebrate development.

Coherent but overlapping expression of microRNAs and their targets during vertebrate development.

Genes & development (2009-02-26)
Alena Shkumatava, Alexander Stark, Hazel Sive, David P Bartel
ABSTRACT

MicroRNAs (miRNAs) are small noncoding RNAs that direct post-transcriptional repression of protein-coding genes. In vertebrates, each highly conserved miRNA typically regulates hundreds of target mRNAs. However, the precise relationship between expression of the miRNAs and that of their targets has remained unclear, in part because of the scarcity of quantitative expression data at cellular resolution. Here we report quantitative analyses of mRNA levels in miRNA-expressing cells of the zebrafish embryo, capturing entire miRNA expression domains, purified to cellular resolution using fluorescent-activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted targets that responded to the miRNAs and distinguished miRNA-mediated mRNA destabilization from other regulatory effects. For all three miRNAs examined, expression of the miRNAs and that of their predicted targets usually overlapped. A few targets were expressed at higher levels in miRNA-expressing cells than in the rest of the embryo, demonstrating that miRNA-mediated repression can act in opposition to other regulatory processes. However, for most targets expression was lower in miRNA-expressing cells than in the rest of the embryo, indicating that miRNAs usually operate in concert with the other regulatory machinery of the cell.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Trypsin-EDTA solution, 1 ×, sterile; sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Sigma-Aldrich
Trypsin-EDTA solution, 10 ×, sterile-filtered, BioReagent, suitable for cell culture, 5.0 g porcine trypsin and 2 g EDTA • 4Na per liter of 0.9% sodium chloride
Sigma-Aldrich
Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Sigma-Aldrich
Trypsin-EDTA solution, 1 ×, sterile-filtered, BioReagent, suitable for cell culture, 500 BAEE units porcine trypsin and 180 μg EDTA • 4Na per ml in Dulbecco′s PBS without calcium and magnesium