TRUE Gene Silencing : Unique Gene Silencing by the tRNA Maturase tRNase ZL under the Direction of Small-guide RNA

Recently, signaling by Wnt and its co-receptor, lipoprotein receptor-related protein (LRP) 5, has been suggested to be involved in the regulation of bone formation and bone mass. However, the mechanism by which Wnt signaling achieves this regulation is currently unknown. To assess the functional contribution of Wnt signaling to osteoblastic differentiation, we generated C2C12 cell lines that overexpress either Wnt3a or Wnt5a and treated these cell lines with bone morphogenetic protein (BMP)-2. Expression of bone matrix protein, matrix extracellular phosphoglycoprotein (MEPE), and matrix metalloproteinase (MMP)-13 was induced by BMP-2 in C2C12 cells overexpressing Wnt3a but not Wnt5a. Functional crosstalk between Wnt and BMP signaling during osteoblastic differentiation was also identified. We have now developed a unique system for downregulating gene expression that involves cutting a specific mRNA using the long form of tRNA 3′ processing endoribonuclease (tRNase ZL) under the direction of a small guide RNA (sgRNA). Any potential off-target effects of sgRNA would be expected to be much less serious than those induced by siRNA because the sgRNA targets complementary RNA of only 7–14 nt, a much smaller length of RNA than that targeted by siRNA. We compared the relative efficiency of the sgRNA/tRNase ZL and RNAi methods using an exogenous luciferase gene target. We found that our new method may be more effective at suppressing gene expression. Here, we also show that tRNaseZL, together with sgRNA, can downregulate expression of the endogenous human genes Bcl-2 and GSK-3β by degradation of their mRNAs. These data suggest that the TRUE (tRNase ZL-utilizing efficacious) gene silencing technology has the potential to be used as a therapeutic agent in the treatment of several diseases.