RNA as a signalling molecule - Intercellular Communication in Plants - page 59

To address this possibility, low-molecular-weight RNAs were selectively enriched
from total RNA fractions of silenced plants and subjected to Northern analysis. It was
found that a discrete species of 21-24 nt long RNAs with antisense sequence of the
silenced genes invariably accumulated in the co-suppressed lines tested (Hamilton
& Baulcombe, 1999). Subsequent studies in Drosophila and C. elegans demon-
strated the involvement of similar molecules in the RNAi process (Tuschl et al. ,
1999; Parrish et al. , 2000).
Interestingly, 21-24 nt long RNAs in the sense orientation were found in equal
abundance in silenced plants, suggesting that these small RNAs accumulated as
duplexes that could be derived from the dsRNA that triggers PTGS/RNAi. This
idea was indeed confirmed by the demonstration that long dsRNA is converted into
21-24 nt RNA duplexes in Drosophila embryo extract (Zamore et al. , 2000). These
duplexes have 5 terminal phosphate and 2 nt long overhanging 3 ends that are char-
acteristic of the product of RNaseIII-like enzymes (Elbashir et al. , 2001c). Analysis
of the Drosophila genome identified three genes that could potentially encode such
proteins. However, only one of these proteins, Dicer, was able to produce 21-24 nt
RNAs when incubated with long dsRNA in embryo extracts (Bernstein et al. , 2001).
Defining features of Dicer, in addition to an RNaseIII domain, include two dsRNA-
binding domains, an RNA helicase domain and a putative protein-protein interaction
domain known as PAZ (PIWI/ARGONAUTE/ZWILLE) (Cerutti et al. , 2000). In
agreement with a key role for Dicer in RNA silencing, genes encoding Dicer-like
enzymes have been identified in the genome of all eukaryotes in which PTGS/RNAi
has been experimentally verified (Hutvagner & Zamore, 2002b). Moreover, some
organisms such as Arabidopsis and Drosophila have several Dicers that may serve
specific functions (Finnegan et al. , 2003). For instance, in silenced plants, siRNAs
with sequence of the silenced gene are in two discrete classes of 21 nt and 24 nt
that are the likely products of separate Dicers and appear to be functionally distinct
(Hamilton et al. , 2002; Tang et al. , 2003; Xie et al. , 2004).
Altogether, these findings support the previously made suggestion that 21-24 nt
RNA duplexes could provide sequence specificity to the machinery that degrades
homologous mRNAs in RNAi/PTGS. In agreement with this suggestion, chemically
synthesized small RNAs with features of Dicer cleavage products were sufficient to
mediate RNAi in Drosophila embryos and in human cells (Elbashir et al. , 2001a,b).
Consequently, the silencing-related small RNAs were coined 'small interfering' or
siRNAs.
3.3.1.4 RNA-induced silencing complex RISC
Fractionation experiments in Drosophila embryo extracts indicated that the nuclease
that generates the siRNAs (Dicer) could be separated from an activity that accounts,
on its own, for the degradation of RNAs that are identical in sequence to the siRNAs
(Hammond et al. , 2000). This activity belongs to a multi-subunit complex referred
to as the 'RNA-induced silencing complex' (RISC), which co-purifies with siRNAs.
RISC activities have been found in other animals, including C. elegans and human
(Hutvagner & Zamore, 2002a; Caudy et al. , 2003). Biochemical characterization of