Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs

Abstract

ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA-and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNAsilencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgenederived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of Ϸ150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.posttranscriptional regulation ͉ ribonuclease ͉ viral RNA ͉ silencing A RGONAUTE (AGO) proteins are implicated in RNAsilencing processes that also involve 21-to 26-nt short RNAs (sRNAs) (1) cleaved from double-stranded or partially doublestranded (ds) RNAs by the RNase III enzyme Dicer. There are several types of RNA-silencing mechanisms, including RNA interference (RNAi), the micro RNA (miRNA) pathway, and RNAdirected chromatin silencing (1). RNAi is a type of RNA silencing in which the Dicer substrate is fully double stranded, the sRNA cleavage product is short interfering RNA (siRNA), and the outcome is targeted destruction of siRNA-complementary RNAs. The miRNA pathway is similar except that the Dicer substrate is an inverted repeat RNA with a partially ds structure, the sRNA is referred to as a miRNA, and the target RNAs can be suppressed at the translational level or degraded as in RNAi, depending on the degree of complementarity between the sRNA and its target. Plants possess an additional class of degradative sRNAs called transacting siRNAs (ta-siRNAs) whose formation depends on the miRNAmediated cleavage of their precursor and its conversion into a dsRNA by RDR6 (2-4). The last pathway, RNA-directed chromatin silencing, is similar to RNAi, but the siRNA targets are either DNA or chromatin-associated RNAs and the outcome is DNA methylation or histone modification at the target locus.In the best understood of these RNA-silencing mechanisms, the duplex siRNAs or miRNAs produced by Dicer are unwound in an ATP-dependent process. One ...

On the origin and functions of RNA-mediated silencing: from protists to man

“…In plants, specific members of the Argonaute subfamily also show distinct activities. Arabidopsis Ago4 has been implicated in DNA and histone methylation whereas Ago1 is an RNA slicer and much more pleotropic in its functions (Baumberger and Baulcombe 2005;Matzke and Birchler 2005;Qi et al 2005;Zilberman et al 2003). Interestingly, in organisms that encode a single Ago-Piwi protein, such as S. pombe and T. brucei, its mutation results in defects in both transcriptional and post-transcriptional silencing (Shi et al 2004;Sigova et al 2004).…”

“…siRNAs are then incorporated into a multiprotein complex, the RNA-induced silencing complex (RISC) (Pham et al 2004;Tomari et al 2004). Members of the Argonaute-Piwi (Ago-Piwi) family of proteins are core components of the RISC and some of these polypeptides function as siRNA-guided endonucleases (Baumberger and Baulcombe 2005;Hammond et al 2001;Liu et al 2004a;Qi et al 2005;Tomari and Zamore 2005). Recent evidence suggests that a siRNA duplex may be loaded into RISC and then Ago cleaves one of the siRNA strands (the passenger strand) triggering its dissociation from the complex (Matranga et al 2005;Miyoshi et al 2005;Rand et al 2005).…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…RISC was first defined as a large RNA-protein complex with sequence-specific RNA cleavage activity that could be purified by chromatographic fractionation from cells programmed in vivo or in vitro with longer dsRNA or small interfering RNA (siRNA) (Hammond et al, 2000;Zamore et al, 2000). Biochemical approaches and genetic screens in protists, plants, fungi and Caenorhabditis elegans have unambiguously identified the members of the Argonaute protein family as essential protein components of both the cleavage-competent RISCs and the miRNAcontaining ribonucleoproteins (Hammond et al, 2001;Mourelatos et al, 2002;Liu et al, 2004;Meister et al, 2004;Baumberger and Baulcombe, 2005;Qi et al, 2005). Argonaute orthologues have been identified in bacteria, archea and most but not all eucaryotes (Cerutti and Casas-Mollano, 2006).…”

A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

“…RNA silencing in protoplasts (1). The mixture is then analyzed by a surface plasmon resonance (SPR) assay for inhibition of the association between RSS and siRNA immobilized on the sensor chip surface (2,3). When any inhibition is detected (3), half of the chemicals in the mixture are tested at a time, repeating this halving process until the candidates are narrowed to a single compound (4).…”