Recognition of Engineered tRNAs with an Extended 3‘ End by Exportin-t (Xpo-t) and Transport of tRNA-Attached Ribozymes to the Cytoplasm in Somatic Cells

Kuwabara, Tomoko; Warashina, Masaki; Sano, Masayuki; Tang, Hengli; Wong‐Staal, Flossie; Munekata, Eisuke; Taira, Kazunari

doi:10.1021/bm0101062

Cited by 26 publications

(14 citation statements)

References 43 publications

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“…Preliminary experiments suggested that a CD4 ϩ T cell line expressing the U5 EGS (560) molecule was protected from infection by a common laboratory strain of HIV-1 at a moi of 0.01 (28). Based on localization studies suggesting a nuclear locale for the RNase P enzyme, we hypothesized that inhibition of viral infection and subsequent cytopathology by the U5 EGS (560) molecule could occur at the level of incoming viral RNA, as well as RNA transcribed from integrated HIV-1 proviral DNA (5,(31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

“…Our PCR results must be tempered with the possibility that real-time PCR may not have the sensitivity to detect a limited number of integrated proviral HIV-1 genomes and that U5 EGS (560) inhibition of viral replication still occurs postintegration at the level of viral genome transcription. Another possibility would have the U5 EGS transcript transported to the cytoplasm of transfected cells following expression by the tRNA promoter and recruiting the RNase P enzyme to inhibit the generation of proviral DNA before reverse transcription of incoming viral RNA (5,33). Alternatively, RNase P-associated EGS molecules may bind to the U5 region of incoming HIV RNA and interfere with the initiation or completion of reverse transcription.…”

Section: Discussionmentioning

confidence: 99%

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Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

Kraus

Geffin

Spruill

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4 ؉ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4 ؉ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection ‫؍‬ 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4 ؉ cells expressing the RNase P-associated U5 EGS (560) molecule.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

Kraus

Geffin

Spruill

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Chimeric tRNAs with 3Ј-attached ribozymes (tRNA-Rzs) have been reported to be efficiently exported to the cytosol in somatic cells, but not in Xenopus oocytes (summarized in Kuwabara et al 2001). Based on exportin-t's substrate specificity (Arts et al 1998b;Lipowsky et al 1999), tRNA-Rz nuclear export would have been anticipated to be exportin-t-independent.…”

Section: Exportin-t/los1p-dependent Trna Nuclear Exportmentioning

confidence: 99%

tRNA transfers to the limelight

Hopper¹,

Phizicky²

2003

Genes Dev.

291

245

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“…1A). The intracellular activities of ribozymes depend on the level of expression of the ribozyme and the secondary structure of the target mRNA in cells (15±20, 24,25). Therefore, we chose to express ribozyme libraries under the control of the promoter of a human gene for tRNA Val , which can be expected to produce high-level expression of transcripts in cells.…”

Section: Resultsmentioning

confidence: 99%

A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries

Kawasaki

Taira²

2002

Nucleic Acids Research

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The sequence of much of the human genome is now available and the next goal is to identify functional genes and to clarify their roles. We have recently developed a novel system for isolation of genes in the Fas-and TNF-a-mediated pathways to apoptosis using poly(A)-connected hammerhead ribozyme libraries with randomized substrate-binding arms at both the 5¢ and 3¢ ends of ribozymes. The transcripts of these hybrid ribozymes have a poly(A) motif that can recruit RNA helicases and, thus, they can effectively attack target sites. In the previous studies, hybrid ribozymes were stably expressed. In order to save selection times, in this study we adopted transiently expressed hybrid ribozymes. In the case of Fas-mediated apoptosis, when we transiently introduced these hybrid-ribozyme libraries into Fasexpressing HeLa cells, we were able to isolate surviving clones that were resistant to or exhibited a delay in Fas-mediated apoptosis. We identi®ed many pro-apoptotic genes and novel genes using this strategy with these transiently expressed hybrid-ribozyme libraries. In contrast, we identi®ed signi®cantly smaller numbers of candidate genes using conventional ribozyme libraries that were expressed transiently. Thus, when changes of a particular phenotype occur within a short period of time, our gene discovery system based on transiently expressed hybrid-ribozyme libraries should also be useful for the rapid identi®cation of functional genes in the post-genome era.

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Recognition of Engineered tRNAs with an Extended 3‘ End by Exportin-t (Xpo-t) and Transport of tRNA-Attached Ribozymes to the Cytoplasm in Somatic Cells

Cited by 26 publications

References 43 publications

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

tRNA transfers to the limelight

A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries

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