Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila.
The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure-function relationship of Dicer, which plays a critical role in the siRNA pathway.
Lim
Do Hwan
DH
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, South Korea.
Kim
Jung
J
Kim
Sanguk
S
Carthew
Richard W
RW
Lee
Young Sik
YS
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D043244:Ribonuclease III; D013329:Structure-Activity Relationship
D029721:Drosophila Proteins; D034741:RNA, Small Interfering; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases