Nucleic Acids Research 2005 33(1):235-243; doi:10.1093/nar/gki164
Published online 12 January 2005
© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
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Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL
Yuichiro Habu2,
Naoko Miyano-Kurosaki1,2,
Michiko Kitano1,
Yumihiko Endo1,
Masakazu Yukita1,
Shigeru Ohira1,
Hiroaki Takaku3,
Masayuki Nashimoto3 and
Hiroshi Takaku1,2,*
1 Department of Life and Environmental Sciences, Chiba Institute of Technology 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
2 High Technology Research Center, Chiba Institute of Technology 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
3 Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences 265-1 Higashito, Niitsu, Niigata 956-8603, Japan
*To whom correspondence should be addressed. Tel: +81 47 478 0407; Fax: +81 47 471 8764; Email: takaku{at}ic.it-chiba.ac.jp
Received December 6, 2004. Revised December 15, 2004. Accepted December 15, 2004.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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