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Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

ABSTRACT: Double-stranded RNA (dsRNA) induces gene-specific silencing in organisms from fungi to animals, a phenomenon known as RNA interference (RNAi). RNAi represents an evolutionarily conserved system to protect against aberrant expression of genes and a powerful tool for gene manipulation. Despite reports that RNAi can be induced in vertebrates, severe sequence-non-specific effects of long dsRNA have been documented in various systems. It has recently been shown in cultured mammalian cells that small interfering RNAs (siRNAs) of 21-23 nt can mediate RNAi but bypass the non-specific response induced by longer dsRNAs. However, the effectiveness of siRNAs has not been demonstrated in living vertebrates. In addition, the mechanism of siRNA suppression of gene expression in vertebrate cells remains to be elucidated. Here we show that synthetic 21 nt siRNAs can specifically inhibit the expression of exogenously introduced as well as endogenous genes in the embryos of Xenopus laevis. siRNAs significantly reduced the steady-state amount of both the mRNA and protein of the cognate gene target. Moreover, co-injection of siRNA with the target RNA transcript specifically suppressed the activity of the latter. Taken together, our findings establish siRNA-mediated post-transcriptional suppression of gene expression in Xenopus embryos.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC101847 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

Zhou Yuan Y Ching Yick-Pang YP Kok Kin Hang KH Kung Hsiang-fu HF Jin Dong-Yan DY

Nucleic acids research 20020401 7

Double-stranded RNA (dsRNA) induces gene-specific silencing in organisms from fungi to animals, a phenomenon known as RNA interference (RNAi). RNAi represents an evolutionarily conserved system to protect against aberrant expression of genes and a powerful tool for gene manipulation. Despite reports that RNAi can be induced in vertebrates, severe sequence-non-specific effects of long dsRNA have been documented in various systems. It has recently been shown in cultured mammalian cells that small ...[more]

PMID: 11917028

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Project description:PurposeMutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. In this study, the feasibility of using small interfering RNAs (siRNAs) to suppress the expression of keratoepithelin and myocilin and their capabilities to reduce the related cytotoxic effects caused by mutant myocilins were investigated.MethodscDNAs of human KE gene and myocilin gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP by KE-specific shRNAs was evaluated by fluorescence microscopy and Western blotting. Suppression of MYOC-EGFP by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of Western blots. A BiP promoter-driven luciferase reporter assay was used to evaluate the stress response of TM cells induced by misfolded mutant myocilins.ResultsTwo KE-specific shRNAs that effectively suppressed the expression of KE-EGFP in HEK293 cells were identified. One shRNA (targeting the coding sequence starting at 1528bp of KE) reduced the expression of KE-EGFP approximately by 50%, whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed greater than 80% of the expression. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of myocilin (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP, ranging from 78% to 90% on average. The activation of BiP gene (as a stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs.ConclusionsKE- or myocilin-specific shRNAs could effectively suppress the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins. RNA interference may have future therapeutic implications in suppressing these genes.

Dataset Information

Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

Publications

Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

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