Gene silencing by the tRNA maturase tRNase ZL under the direction of small-guide RNA

Abstract

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3 0 -processing endoribonuclease (tRNase Z L ) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z L in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z L method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z L directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z L together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3b by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells

“…Michigan, Ann Arbor, MI) (Yu et al, 2003a). The sgRNA expression plasmids for silencing GSK-3β genes (pRNA Tin-H1.2/Neo-sgGSKL) were constructed as described previously (Nakashima et al, 2007). pU6pro (Yu et al, 2003a) or pRNATin-H1.2/Neo-Luc (Nakashima et al, 2007) was used as a non-targeting control.…”

“…The sgRNA expression plasmids for silencing GSK-3β genes (pRNA Tin-H1.2/Neo-sgGSKL) were constructed as described previously (Nakashima et al, 2007). pU6pro (Yu et al, 2003a) or pRNATin-H1.2/Neo-Luc (Nakashima et al, 2007) was used as a non-targeting control. The plasmid DNA was transfected into cells using the cationic lipid reagent Lipofectamine PLUS (Invitrogen, Carlsbad, CA) as described previously (Nakashima et al, 2005).…”

“…Since GSK-3β is known to mediate canonical Wnt signaling, we examined the effect of knockdown of GSK-3β using either RNA interference or tRNase Z L -utilizing efficacious gene silencing (TRUE gene silencing) (Tamura et al, 2003, Nakashima et al, 2007, Elbarbary et al, 2009) upon CXCL12 induction.…”

Human cytosolic tRNase ZL can downregulate gene expression through miRNA

“…The present results together with this observation imply that other types of small cellular ncRNA would also mediate regulation of gene expression by tRNase Z L . It is now clear that the reason why the TRUE gene silencing method works well in the cells [14][15][16][17] is because it utilizes artificial sgRNAs like naturally occurring ncRNAs and mimics the intrinsic cellular gene regulatory system. …”

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing

“…The efficacy of TRUE gene silencing has been demonstrated by using it to introduce various artificially-designed sgRNAs into living cells either by their expression plasmids or by 2'-O-methyl RNAs (2,7). sgRNA is divided into four groups: 5'-half-tRNA, RNA heptamer, hook RNA and ~14-nt linear RNA (1,2,8,9).…”