RNA Processing Activities of the Arabidopsis Argonaute Protein Family

Abstract

Stringent regulation of gene expression is essential for all organisms, and eukaryotes rely on diverse RNA silencing mechanisms for this regulation at both the transcriptional and posttranscriptional level. For example, transcriptional gene silencing (TGS) maintains genome integrity by controlling the replication of transposons and other repetitive DNA elements, as well as preserving chromatin states and epigenetic imprinting. Post-transcriptional gene silencing (PTGS) mechanisms on the other hand control the expression of messenger RNA (mRNA) transcripts of protein-coding genes in order to regulate developmental transitions and responses to environmental stresses. In plants, both transcriptional and posttranscriptional RNA silencing mechanisms are also involved in the defence against invading pathogens, especially viruses. RNA silencing pathways are directed by a specific class of small RNA (sRNA) which are predominantly 20 to 25 nucleotides (nt) in length. These sRNAs are processed from longer precursor molecules of either perfectly or imperfectly double-stranded RNA (dsRNA) by a member of the DICER RNase III-like endonuclease family (Bernstein et al., 2001; Gregory et al., 2005). Once processed from its dsRNA substrate, the sRNA is subsequently modified and loaded into an RNaseH-like Argonaute (AGO) protein to form the catalytic core of an RNA-induced silencing complex (RISC). RISC uses the loaded sRNA as a sequencespecificity guide to direct RNA silencing of the targeted sequence at either the transcriptional or post-transcriptional level depending on; i) the class of sRNA loaded by AGO, and; ii) the AGO protein family member loaded with the sRNA. Although the effector function mediated by the sRNA-loaded AGO protein is highly conserved amongst eukaryotes, the number of AGO proteins encoded by different species varies widely (Tolia & Joshua-Tor, 2007; Hutvagner & Simard, 2008; Vaucheret, 2008). For example, Caenorhabditis elegans (C. elegans) has twenty seven AGOs, Drosophila melanogaster (Drosophila) five, humans four and the yeast Schizosaccharomyces pombe one. In Arabidopsis thaliana (Arabidopsis), the model dicotyledonous plant species, the AGO protein family consists of ten members, that mediate the parallel RNA silencing pathways of Arabidopsis, and which are directed by numerous classes of endogenous sRNA, including the microRNA (miRNA; Lee & Ambros, 2001), small-interfering RNA (siRNA; Hamilton & Baulcombe, 1999), repeat-associated small-interfering RNA (rasiRNA; Meister & Tuschl, 2004), transacting small-interfering RNA (tasiRNA; Adenot et al., 2006; Xie et al., 2005) and natural antisense transcript small-interfering RNA (natsiRNA; Borsani et al., 2005) classes of sRNA.