RNA as a signalling molecule - Intercellular Communication in Plants - page 60

the Drosophila RISC revealed that it contains several proteins, among which is Arg-
onaute 2 (Ago2), a homologue of the translation initiation factor eIF2C (Hammond
et al. , 2001). Interestingly, Ago2 contains a PAZ domain that is potentially involved
in mediating Dicer-RISC interaction. Upon this interaction, it is thought that the
Dicer-generated siRNAs are incorporated into RISC and thereby guide degradation
of homologous RNAs in a sequence-specific manner. This scenario also most likely
applies in plants, since a RISC activity has been detected in wheat germ extracts
(Tang et al. , 2003). The endonucleolytic cleavage of RISC targets occurs at the
centre of the siRNA/RNA hybrid (11th nucleotide) (Elbashir et al. , 2001c) and is
mediated by Ago2 in Drosophila (Liu et al. , 2004; Meister et al. , 2004).
3.3.1.5 Transitive RNA silencing
As explained above, cellular RdRps play a key role in the initiation of sense trans-
gene PTGS in plants and of quelling in N. crassa , presumably through recognition
of transgene-derived aberrant RNAs and their subsequent conversion into double-
stranded molecules. In addition, cellular RdRps may also play important functions in
amplifying already established silencing. For instance, it was shown, in C. elegans ,
that RNAi triggered by siRNAs corresponding to one part of a transcript leads to
production of secondary siRNAs that are homologous in sequence to regions lo-
cated outside of the initially targeted portion of this transcript (Sijen et al. , 2001).
This process leading to de novo synthesis of siRNAs is referred to as 'transitiv-
ity' and requires the action of RFF1, a putative RdRp homologue of C. elegans
(Sijen et al. , 2001). It was proposed that the primary siRNAs that trigger RNAi
prime the activity of RRF1 such that it converts siRNA-homologous transcripts into
dsRNA that are subsequently cleaved by Dicer into secondary siRNAs. The idea of
this 'primer-dependent' transitivity was supported by the fact that only secondary
siRNAs located 5 of the initially targeted region were apparently synthesized (Sijen
et al. , 2001). Thus, by generating more siRNAs than originally used to trigger RNAi,
the RFF1-mediated transitivity may account for the very high potency of RNAi in
C. elegans (Fire et al. , 1998).
Transitivity had also been reported in transgenic tobacco exhibiting PTGS and
in Arabidopsis infected with recombinant viruses or expressing constitutively an
inverted repeat transgene (Voinnet et al. , 1998; Vaistij et al. , 2002; Himber et al. ,
2003). Although, as in C. elegans , this process was dependent upon the activity of
an RdRp (SDE1), occurrence of secondary siRNAs was from both the 5 and 3 part
of the originally targeted transcript (Voinnet et al. , 1998; Vaistij et al. , 2002). To
account for this phenomenon, it is possible that 5 transitivity is primer-dependent
but that 3 transitivity is primer-independent in plants. For instance, it has been
suggested that the 3 cleavage products generated by the action of RISC could
be perceived as 'aberrant' and used directly as templates by SDE1 (Hutvagner &
Zamore, 2002b), in a manner similar to the events that initiates co-suppression and
quelling from sense transcripts (see above section). In any case, and as seen in
C. elegans , the involvement of transitivity in RNA silencing in plants provides a