In vitro synthesized small interfering RNAs elicit RNA interference in african trypanosomes: an in vitro and in vivo analysis.
RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific double-stranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.
Best
Alexander
A
Department of Microbiology and Genetics, Darmstadt University of Technology, 64287 Darmstadt, Germany.
Handoko
Lusy
L
Schlüter
Elke
E
Göringer
H U
HU
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D043244:Ribonuclease III / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014404:Tubulin / Q000235:genetics
D002413:Cations, Divalent; D012333:RNA, Messenger; D016053:RNA, Protozoan; D034741:RNA, Small Interfering; D014404:Tubulin; D000255:Adenosine Triphosphate; D043244:Ribonuclease III