Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants.
RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.
Wang
Ming-Bo
MB
Commonwealth Scientific and Industrial Research Organisation Plant Industry, Canberra, Australian Capital Territory 2601, Australia. ming-bo.wang@csiro.au
Helliwell
Christopher A
CA
Wu
Li-Min
LM
Waterhouse
Peter M
PM
Peacock
W James
WJ
Dennis
Elizabeth S
ES
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019175:DNA Methylation; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D017343:Genes, Plant; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D018749:RNA, Plant / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D018744:DNA, Plant; D018749:RNA, Plant; D034741:RNA, Small Interfering; D012319:RNA Polymerase II; D012320:RNA Polymerase III