Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL

Abstract

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

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

“…Taken together, these results suggest that gene-silencing efficiency of sgRNA would generally Gene silencing by tRNase Z L and sgRNA A Nakashima et al become comparable to that of siRNA as an sgRNA amount is increased. tRNase Z L is responsible for the gene silencing by sgRNA Although these and previous results 15,16 indicated that appropriate sgRNAs can downregulate targeted gene expressions in the cells, no direct evidence existed demonstrating that the downregulation is owing to specific mRNA cleavage by endogenous tRNase Z L not owing to a simple antisense effect or other different mechanisms. To clarify this issue, we generated two types of stable 293 cell line, which produce more tRNase Z L or less tRNase Z L and examined silencing levels for the luciferase gene in these transfectants.…”

“…4,6 We have shown the efficacy of this RNA-targeting method in the living cells to some degree by introducing sgRNAs either as their expression plasmids or as 2 0 -Omethyl RNAs. 15,16 The expression of exogenous reporter genes for Escherichia coli chloramphenicol acetyltransferase (CAT) and firefly luciferase were downregulated by appropriately designed sgRNAs in mammalian culture cells, 15 and the HIV-1 expression in Jurkat cells was almost completely suppressed by a 5 0 -half-tRNA-type sgRNA up to 18 days. 16 However, the involvement of tRNase Z L in this gene silencing remained unproven.…”

Suppression of HIV-1 Replication by a Combination of Endonucleolytic Ribozymes (RNase P and tRNase ZL)

“…Mo-MLV-based sgRNA-SL4 targeting the HIV-1 gag gene could suppress sgRNA-dependent HIV-1 expression in human T cells. [23] In this article, we demonstrate the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two truncated short extra guide sequences (sEGS) specifically recognize the tat and vif regions of HIV-1 mRNA and mediate subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components.…”

“…Furthermore, we demonstrated the inhibition of HIV-1 gene products in cultured cells by inducing HIV-1 mRNA cleavage using a modified 5 -half-tRNA Arg (sgRNA) and mammalian tRNase ZL. [23] The greatest inhibitory effect on HIV-1 expression was achieved using sgRNA targeting the HIV-1 gag gene.…”

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. …”