Transcriptional and posttranscriptional gene silencing are mechanistically related.
Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.
Sijen
T
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Department of Developmental Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV, The, Amsterdam, Netherlands.
Vijn
I
I
Rebocho
A
A
van Blokland
R
R
Roelofs
D
D
Mol
J N
JN
Kooter
J M
JM
D000217:Acyltransferases / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D020868:Gene Silencing; D017343:Genes, Plant; D006836:Hydro-Lyases / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded; D018749:RNA, Plant; D019657:Solanaceae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D012330:RNA, Double-Stranded; D018749:RNA, Plant; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; D000217:Acyltransferases; C020141:flavanone synthetase; D006836:Hydro-Lyases; C070395:mannopine synthase