Small interfering RNAs are generally defined as small RNAs that silence transcripts from which they originate (Bartel, 2004). They were first described in plants, where it was shown that the silencing of three transgenes involved a small antisense RNA c. 25 nt long complementary to each targeted mRNA (Hamilton & Baulcombe, 1999; Hamilton et al., 2002; Tang et al., 2003). In plants, siRNAs have a variety of functions that can be grouped in at least two broad categories: those that trigger changes in the chromatin state of elements from which they derive and those that derive from and defend against exogenous RNA sequences such as viruses or sense transgene transcripts (for review see Baulcombe, 2004).
1. Small interfering RNA silencing of exogenous dsRNA sequences
The siRNAs and miRNA sequences are targeted to a complex called RNA-induced silencing complex (RISC; Fig. 2). Argonaute (AGO) proteins are a core component of this complex (Hammond et al., 2000; Nykanen et al., 2001; Schwarz et al., 2003; Pham et al., 2004; Tomari et al., 2004). In Arabidopsis, AGO1 (Bohmert et al., 1998) is associated with miRNAs, trans-acting siRNAs and transgene-derived siRNAs but not with virus-derived siRNAs and siRNAs involved in chromatin silencing (Fagard et al., 2000; Boutet et al., 2003; Vaucheret et al., 2004; Kidner & Martienssen, 2005). Mutants of AGO1 were shown to be hypersensitive to virus infections (Morel et al., 2002). Some transposons were shown to be upregulated in ago1 mutants (Lippman et al., 2003). It was also shown that at least in vitro, AGO1 does not seem to have other partners in RISC and would be solely interacting with small RNAs, unlike what is observed in animals (Baumberger & Baulcombe, 2005).
Figure 2. Small RNA pathways in plants. (a) Plant microRNA (miRNA) biogenesis. MicroRNA genes are transcribed from their own locus by POL-II. The hairpin-like secondary structure is further processed by DICER in several steps to produce miRNA:miRNA* duplexes. The duplexes are then methylated by HEN1, before being exported to the cytoplasm, possibly by HASTY. Here the duplex is unwound and the miRNA is associated with AGO1. This complex, known as RISC, will bind specifically to a target messenger RNA, and guide its cleavage (in most of the cases) or will repress its translation. DCL, Dicer-like; HYL, HYPONASTIC LEAVES. (b) Small interfering RNAs (siRNAs). Long double-stranded RNAs (dsRNAs) from diverse origins (viruses, transpososons, transgenes, etc.) are converted into 21 nucleotide (nt) long siRNAs by DICER enzymes. These small RNAs are then loaded into RISC and associated with AGO4 or another Argonaute protein. The complex will then bind to the same messenger RNA from which they originate, and cleave the mRNA, silencing its expression. Small interfering RNAs can also bind to the mRNA and initiate the transformation of single-stranded RNA (ssRNA) into dsRNA, thus amplifying siRNA production. RDR, RNA-dependent RNA polymerase. (c) trans-Acting siRNAs. In plants, some miRNAs (miR173 and miR390) cleave a target mRNA expressed from ta-siRNA loci. After cleavage, either the 5′- or the 3′-terminus is converted into dsRNA by RDR enzymes, and then processed into 21 nt siRNAs that guide degradation of target mRNA that is different from the ta-siRNA transcript from which they originated.
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The RNA-dependent RNA polymerases (RDRs) RDR1 and RDR6 are required in the siRNA pathway that silences viruses and transgenes (Dalmay et al., 2000; Mourrain et al., 2000; Xie et al., 2001). Those proteins turn single-stranded (ss) RNA into dsRNA, with or without a siRNA as a primer (Baulcombe, 2004). As a result of the action of RDRs, a single RNA or primary siRNA molecule can generate many dsRNA, thus amplifying the response (Fig. 2b). Unexpectedly, it was shown that RDR6 might also repress the expression of a miRNA, miR165/166 (Li et al., 2005).
The dsRNA structures are further processed by a member of the Dicer family that generates small RNAs from double-stranded RNA sequences with 2-nt overhangs at the 3′ ends (Bernstein et al., 2001). In Arabidopsis, four Dicer-like (DCL) proteins are known, each one having a distinct function in different small RNA pathways (Schauer et al., 2002; Xie et al., 2004; Dunoyer et al., 2005; Gasciolli et al., 2005; Xie et al., 2005b). DCL1 produces miRNAs, DCL2 produces siRNAs involved in the silencing of at least some viral sequences (Xie et al., 2004) and DCL3 produces siRNAs involved in DNA methylation and heterochromatin formation (Xie et al., 2004). Recent work suggests that DCL4 produces siRNAs triggered by inverted-repeat transgenes in plants (Dunoyer et al., 2005) and is also associated with the ta-siRNA pathway (Gasciolli et al., 2005; Xie et al., 2005b; Yoshikawa et al., 2005). Some results have also shown that a partial functional redundancy amongst the different Dicer-like proteins in Arabidopsis is possible (Gasciolli et al., 2005; Xie et al., 2005b).
Virus dsRNA sequences are recognized by the RNA silencing machinery, which produces siRNAs that will silent viral genes and prevent the accumulation of the pathogen (for review see Dunoyer & Voinnet, 2005). Virus defense via siRNA is likely to be an ancient mechanism and therefore viruses have evolved various ways to bypass this barrier (Baulcombe, 2004; Dunoyer & Voinnet, 2005; Simon-Mateo & Garcia, 2006). The mechanism best known involves the production of a protein by the virus that will block the silencing pathway of the host (Voinnet et al., 1999; Mallory et al., 2002; Kasschau et al., 2003; Ye et al., 2003; Chapman et al., 2004; Dunoyer et al., 2004; Lakatos et al., 2004). But other mechanisms might also exist. In plants, the expression of siRNAs from inverted-repeats transgenes mimic the symptoms observed during viroid infections, suggesting RNA silencing of the host genes (Wang et al., 2004b). Conversely, many Arabidopsis siRNAs do not show a high degree of similarity to any Arabidopsis mRNA. Therefore, one interesting hypothesis is that they could constitute a reservoir of defense molecules because of their complementarity to viral sequences (Dunoyer & Voinnet, 2005). This RNA silencing defense might not be limited to viruses: it was also shown recently that bacterial infection by a virulent Agrobacterium tumefaciens triggered a rather complex siRNA-mediated silencing response (Dunoyer et al., 2006).
Another striking feature of siRNA-mediated silencing in plants and in some animals is its systemic nature: the effect of silencing can extend beyond the site of initiation and spread through the organism (for a review see Voinnet, 2005). Recent work suggests that DCL4 is responsible for the production of the 21-nt long siRNAs involved in the cell-to-cell silencing signal (Dunoyer et al., 2005). The movement of siRNAs or miRNAs could be important for the regulation of endogenous genes. For example, it is known that the distribution of miR165/166 in the leaf, where they act as repressors of genes affecting leaf polarity, resembles that of a mobile signal (Emery et al., 2003; Juarez et al., 2004; Kidner & Martienssen, 2004). Many siRNAs and miRNAs were detected in the phloem sap of pumpkin, where a protein has been characterized that binds specifically to small RNAs (Yoo et al., 2004).
Recently, a study revealed that an antisense overlapping gene pair generated two types of siRNAs involved in salt-stress tolerance (Borsani et al., 2005). Those genes are P5CDH, a stress-related gene, and SRO5, a gene of unknown function. When both transcripts are present, a 24-nt siRNA is formed by a biogenesis pathway dependent on DCL2, RDR6, SGS3 and NRPD1A. The cleavage of the P5CDH transcript by the 24-nt siRNA then sets the phase for the generation of 21-nt siRNAs by DCL1 that will further cleave the P5CDH transcript. The expression of SRO5 is induced by salt, a step thus necessary for the initial siRNA formation. This elegant study shows that endogenous siRNAs (dubbed nat-siRNAs), derived from a pair of natural cis-antisense transcripts, regulate salt tolerance. Given that overlapping genes are not rare in many eukaryotic genomes, nat-siRNA-based regulation might also occur in many other processes (Borsani et al., 2005).
2. Small interfering RNAs and chromatin state
Experiments have shown that expressed siRNAs were matching transposable element sequences that could form imperfect RNA duplexes (Mette et al., 2002). More recently, high-throughput sequencing of expressed small RNAs in Arabidopsis (Lu et al. 2005a) showed that many siRNAs are associated with transposons silenced by methylation. Maintaining this silenced state involves a low level of transcription, which is a paradox because silencing inhibits transcription by known DNA-dependent RNA polymerases (Lippman & Martienssen, 2004). However, the recent finding of a new RNA polymerase might help to solve this issue. Two studies have described a new RNA polymerase named POL-IV that directs heterochromatic silencing, although the mechanism is not yet clear (Herr et al., 2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005; Vaughn & Martienssen, 2005). The RNA-dependent RNA polymerase RDR2 was shown to be required for heterochromatin formation, as well as the Dicer-like protein DCL3 and the Argonaute protein AGO4 (Zilberman et al., 2003; Xie et al., 2004; Zilberman et al., 2004).
Unexpectedly, similarly to siRNAs, miRNAs might also contribute to DNA methylation in Arabidopsis. There is experimental evidence that mir165/166, which targets the PHAVOLUTA (PHV)- and PHABULOSA (PHB)-encoding mRNAs, induces methylation of PHV and PHB genes downstream of the miRNA target sites (Bao et al., 2004). MicroRNA pairing very likely takes place with the nascent but already spliced transcript, implying that miRNAs may also be active in the nucleus, at least in plants.