An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.
RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.
Shi
Huafang
H
Department of Internal Medicine, Yale University Medical School, New Haven, Connecticut 06536-0812, USA.
Tschudi
Christian
C
Ullu
Elisabetta
E
D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins; D053487:DEAD-box RNA Helicases; D004722:Endoribonucleases; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
C495987:AGO1 protein, Trypanosoma brucei; D060565:Argonaute Proteins; D015800:Protozoan Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D004722:Endoribonucleases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases