RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase.
We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct that is transcribed into a hairpin RNA. Gene silencing was accompanied by the appearance of sequence-specific RNA about 23mers and seemed to have a limited capacity. The three Dictyostelium homologues of the RNA-directed RNA polymerase (RrpA, RrpB, and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RNA-directed RNA polymerase and the dicer homologue. Only the knock-out of rrpA resulted in a loss of the hairpin RNA effect and simultaneously in a loss of detectable about 23mers. However, about 23mers were still generated by the Dictyostelium dsRNase in vitro with extracts from rrpA(-), rrpB(-), and DosA(-) cells. Both RrpA and a target gene were required for production of detectable amounts of about 23mers, suggesting that target sequences are involved in about 23mer amplification.
Martens
Henrik
H
Abt. Genetik, Universität Kassel, D-34132 Kassel, Germany.
Novotny
Jindrich
J
Oberstrass
Jürgen
J
Steck
Theodore L
TL
Postlethwait
Pamela
P
Nellen
Wolfgang
W
D000818:Animals; D004023:Dictyostelium / Q000235:genetics*; D000070821:Discoidins; D020868:Gene Silencing; D037102:Lectins; D015800:Protozoan Proteins / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D012260:Ribonucleases / Q000235:genetics*
D000070821:Discoidins; D037102:Lectins; D015800:Protozoan Proteins; D012330:RNA, Double-Stranded; D012324:RNA-Dependent RNA Polymerase; D012260:Ribonucleases