Generation and Application of Asymmetric Hammerhead Ribozymes

Hammann, Christian; Tabler, Martin

doi:10.1006/meth.1999.0784

Cited by 7 publications

(1 citation statement)

References 35 publications

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“…The expression vector encoding the rat HNF4-A-(227-455) and rat HNF4-A-(⌬271-354) proteins was the generous gift of Dr. Iannis Talianidis (IMBB-FORTH, Heraklion). The design of the antisense ribozyme construct pCDNA1-HNF-4 Rz was described previously (30,31). The expression vector encoding the 6ϫ Myc-tagged human HNF-4 protein (pCDNA1-6ϫMyc-HNF4) was constructed by subcloning the fulllength human HNF-4 cDNA from pMT2-hHNF-4 (15) to the EcoRI site of pCDNA1-6ϫMyc vector.…”

Section: Methodsmentioning

confidence: 99%

SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

Kardassis

Pardali²,

Zannis

2000

Journal of Biological Chemistry

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Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15-70-fold) the ؊890/؉24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the ؊890/؉24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor ␤ and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.

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Section: Methodsmentioning

confidence: 99%

SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

Kardassis

Pardali²,

Zannis

2000

Journal of Biological Chemistry

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Ribozyme-Mediated Suppression of G Protein γ Subunits

Robishaw¹,

Wang²,

Schwindinger³

2002

G Protein Pathways, Part B: G Proteins and Their Regulators

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Length Variation of Helix III in a Hammerhead Ribozyme and Its Influence on Cleavage Activity

Hammann

Martı́nez²,

Moosbauer³

et al. 1999

Antisense and Nucleic Acid Drug Development

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The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.

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Generation and Application of Asymmetric Hammerhead Ribozymes

Cited by 7 publications

References 35 publications

SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

Ribozyme-Mediated Suppression of G Protein γ Subunits

Length Variation of Helix III in a Hammerhead Ribozyme and Its Influence on Cleavage Activity

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