The Effect of MicroRNA-31 on the small GTPase Arf6 and the role of Arf6 in Early Embryogenesis
Date
2013-05
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Publisher
University of Delaware
Abstract
microRNAs (miRNAs) are small, non-coding mRNAs that repress protein translation and/or mRNA degradation. Our lab has demonstrated that microRNAs are vital for early development. microRNA-31 is one of the most abundant miRNAs in the sea urchin embryo, and it is essential for proper cell specification. We bioinformatically identified Arf6 to be a potential target of microRNA-31 and found an increase in Arf6 protein levels in microRNA-31 depleted embryos, suggesting that Arf6 may be regulated by microRNA-31. While the function of Arf6 has been studied extensively at the cellular level using human tissue culture, its regulatory function in early development has not been examined in detail. Arf6 is a small GTPase protein that mediates membrane trafficking between the plasma membrane and endosomal compartments. It also mediates receptor endocytosis and actin rearrangement. We hypothesize that Arf6 is regulated by microRNA-31 and directs cell movement and specification of mesenchymal cells. Quantitative, real time PCR and western blot analysis revealed that the Arf6 mRNA expression peaks at the 32-cell stage and that Arf6 protein levels increase during gastrulation, respectively. We have tested the function of Arf6 with morpholino antisense oligonucleotides, constitutively active (Q67L), and dominant negative (T27N) Arf6 mutants. The Arf6 knockdown phenotypes include thickened filopodia, exogastrulation and vegetal cell detachment, indicating that Arf6 is critical for proper early development. The Arf6 mutant phenotypes include widened blastopore, altered gut and disruption of the arrangement of primary mesenchyme cell and endodermal cells. This research provides insight into the function of Arf6 during early embryogenesis and its regulation by miRNA-31 during early development.