Redesigning Animal Agriculture

128 T. Doran and L. Lambeth
ATP
ADP + Pi
5�-P
3�-HO
ATP
ADP + Pi
dsRNA
(Bernstein et al., 2001), and is evolutionarily
conserved in worms, flies, fungi, plants
and mammals. A proposed model for Dicer
involves the ATP-dependent translocation of
the enzyme along its target prior to dsRNA
cleavage, the efficiency of which has been
shown to be directly proportional to the
length of the target (Bernstein et al., 2001).
Dicer has four distinct domains: an aminoterminal
helicase domain, dual RNAse III
motifs, a dsRNA binding domain, and a PAZ
domain (named after its component proteins
Piwi, Argo, and Zwile/Pinhead) (Tabara
et al., 1999; Catalanotto et al., 2000).
Following the cleavage of dsRNA into
siRNAs by Dicer the second important stage
OH-3�
P-5� siRNA duplex
RISC
RISC activation and
siRNA unwinding
RISC
Dicer
RISC-mediated
target recognition
mRNA cleavage
and degradation
Fig. 8.1. The cellular mechanism of RNAi. Endogenous dsRNA from repetitive sequences such as
transposons and exogenous dsRNA from viral origin trigger the RNAi pathway. These dsRNA substrates
are processed by Dicer into siRNA duplexes with 3’ overhangs and 5’-phosphate and 3’-hydroxyl termini.
The siRNA then binds to the multiprotein complex to form the RISC. Activation of RISC is accompanied by
siRNA unwinding of the siRNA duplex, and the antisense strand is guided to the target mRNA. Binding of
the homologous siRNA strand results in the RISC-mediated cleavage of the target mRNA.
of mRNA degradation involves the form ation
of a ribonucleic protein complex termed
RNA-induced silencing complex (RISC),
which guides the siRNAs to their target
RNAs (Fig. 8.1). The process is very similar
to the processing of miRNAs, except that
the miRNA precursors are first processed in
the nucleus by an RNAse III enzyme termed
Drosher prior to Dicer cleavage, and the pairing
of processed miRNA to its target RNA by
RISC often contains mismatches, which leads
to translational repression of target RNAs
(Doench et al., 2003; Bartel and Chen, 2004).
RISC was originally identified based
on its requirement for siRNA binding for
gene silencing in cultured Drosophila cells