Copyright Ó 2008 by the Genetics Society of America DOI: 10.1534/genetics.107.086546 Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing J. Armando Casas-Mollano,1 Jennifer Rohr,1 Eun-Jeong Kim, Eniko Balassa, Karin van Dijk2 and Heriberto Cerutti3 School of Biological Sciences and Plant Science Initiative, University of Nebraska, Lincoln, Nebraska 68588 Manuscript received December 28, 2007 Accepted for publication March 5, 2008 ABSTRACT Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER- LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga. NA-MEDIATED silencing is an evolutionarily con- Aoki et al. 2007; Aravin et al. 2007; Matranga and Zamore R served process by which double-stranded RNA 2007; Pak and Fire 2007). Intriguingly, recent results (dsRNA) induces the inactivation of cognate sequences indicate that small RNAs not only function in gene silenc- through a variety of mechanisms, including translation ing but also may participate in activation of expression (Li inhibition, RNA degradation, transcriptional repres- et al. 2006; Janowski et al. 2007; Vasudevan et al. 2007). sion, or DNA elimination (Baulcombe 2004; Meister Genetic and biochemical studies from multiple organ- and Tuschl 2004; Matzke and Birchler 2005; isms have led to the identification of three core compo- Matranga and Zamore 2007). dsRNA-triggered silenc- nents of the RNAi machinery, namely Dicer, Argonaute- ing was initially characterized in Caenorhabditis elegans Piwi (AGO-Piwi), and RNA-dependent RNA polymerase and termed RNA interference (RNAi) (Fire et al. 1998), (RDR) (Cogoni and Macino 2000; Baulcombe 2004; but this phenomenon is now known to occur in a wide Zamore and Haley 2005). Long or hairpin dsRNAs are spectrum of eukaryotes (Cerutti and Casas-Mollano processed into small RNAs by the RNaseIII-like endo- 2006). Moreover, despite the mechanistic diversity of nuclease Dicer (Bernstein et al. 2001; Meister and RNA-mediated repression, all characterized pathways Tuschl 2004; Zamore and Haley 2005). These small appear to involve small RNAs (20–30 nucleotides in RNAs are then incorporated into effector complexes, length) generated by the processing of dsRNAs, with the which include members of the AGO-Piwi family of possible exception of Piwi-associated RNAs and certain proteins. This family consists of two main classes of small RNAs produced directly by polymerization (Bartel polypeptides, one named after Arabidopsis thaliana AR- 2004; Brodersen and Voinnet 2006; Vaucheret 2006; GONAUTE1 and the other after Drosophila melanogaster Piwi (Carmell et al. 2002; Cerutti and Casas-Mollano ravin Sequence data from this article have been deposited with the EMBL/ 2006; A et al. 2007). AGO-Piwi proteins contain two GenBank Data Libraries under accession no. EU368690. conserved motifs: the PAZ (P iwi/Argonaute/Zwille) 1These authors contributed equally to this work. domain, which binds to the 39-ends of small RNAs, and 2Present address: Department of Biology, Creighton University, Omaha, the Piwi domain, which is structurally related to RNase H NE 68178. (Cerutti et al. 2000; Ma et al. 2004; Song et al. 2004; Yuan 3Corresponding author: School of Biological Sciences and Plant Science Initiative, University of Nebraska, E211 Beadle Center, Lincoln, NE et al. 2005). Some AGO-Piwi polypeptides function as 68588-0666. E-mail: [email protected] small RNA-guided endonucleases that cleave comple- Genetics 179: 69–81 (May 2008) 70 J. A. Casas-Mollano et al. mentary RNAs (Liu et al.2004;Meister et al. 2004; Unicellular eukaryotes appear to have fewer RNAi Baumberger and Baulcombe 2005). Others, in contrast, components and small RNA-mediated pathways than are not endonucleolytically active (Liu et al. 2004; multicellular ones (Cerutti and Casas-Mollano 2006). Meister et al. 2004; Rivas et al. 2005) and may be part Yet many unicellular organisms also possess relatively of effector complexes involved in nondegradative RNAi small nuclear genomes and therefore it is not clear processes (Wienholds and Plasterk 2005; Zamore and whether diversification of RNA-mediated silencing is a Haley 2005). In certain organisms such as nematodes, unique innovation coupled to the evolution of multi- land plants, and fungi, RDRs also play an important role cellularity or whether it is associated mainly with com- in RNAi (Cogoni and Macino 2000; Sijen et al.2001; plex genomes, regardless of cellularity, where the Baulcombe 2004). In these species, RDR activity may additional level of regulation may confer a selective initiate RNAi by producing dsRNA from single-stranded advantage. In this respect, the unicellular green alga transcripts or dramatically enhance the RNAi response by Chlamydomonas reinhardtii may provide valuable insights amplifying the amounts of small RNAs (Cerutti 2003; since its 120-Mb nuclear genome (Merchant et al. Baulcombe 2004; Aoki et al.2007;Pak and Fire 2007). 2007) is similar in size to that of the higher plant A. In multicellular eukaryotes, duplication and diversi- thaliana. Chlamydomonas possesses a functional RNAi fication of the protein components for small RNA bio- machinery, as reflected by the generation of siRNAs genesis and/or of those involved in effector functions from inverted repeat transgenic transcripts and the have resulted in complex, partly overlapping pathways corresponding suppression of expression of target for the epigenetic control of gene expression (Okamura genes (Rohr et al. 2004; Ibrahim et al. 2006; Schroda et al. 2004; Matzke and Birchler 2005; Brodersen 2006). Moreover, this alga has recently been shown to and Voinnet 2006; Vaucheret 2006; Chapman and contain a complex set of small RNAs including miRNAs, Carrington 2007; Matranga and Zamore 2007). phased siRNAs, as well as siRNAs originating from Three major classes of small RNAs have been identified transposons and repeats (Molnar et al. 2007; Zhao in animals: microRNAs (miRNAs), Piwi-interacting RNAs et al. 2007). Yet the RNAi machinery and its biological (piRNAs), and small interfering RNAs (siRNAs) (Aravin role(s), in particular with regard to pathway specializa- et al. 2007; Matranga and Zamore 2007). Plant species tion, have not been explored in detail in Chlamydomo- lack Piwi proteins (Cerutti and Casas-Mollano 2006; nas. Here we examine the core RNAi components Aravin et al. 2007) and contain only miRNAs and siRNAs. encoded in the C. reinhardtii genome and demonstrate MicroRNAs originate from endogenous RNA transcripts that DCL1 is involved in the post-transcriptional silenc- that fold into imperfect stem-loop structures. They often ing of the TOC1 retrotransposon. This function is not modulate the expression of genes with roles in de- compensated by two other Dicer paralogs, indicating velopment, physiological processes, or stress responses that RNAi pathway diversification does occur in Chla- (Bartel 2004; Wienholds and Plasterk 2005; Chapman mydomonas. However, when grown under standard and Carrington 2007; Matranga and Zamore 2007). laboratory conditions, this alga relies primarily on a piRNAs are longer than miRNAs or siRNAs and interact transcriptional silencing mechanism(s) to control mo- specifically with Piwi proteins, and some of them seem bile genetic elements. In fact, C. reinhardtii appears to to function in the control of mobile genetic elements have several, at least partly independent, transposon (Aravin et al. 2007; Matranga and Zamore 2007). Their silencing pathways that operate at either the transcrip- biogenesis is not clearly understood but piRNAs appear to tional or the post-transcriptional levels. derive from single-stranded transcripts by a Dicer-inde- pendent mechanism (Aravin et al. 2007; Matranga and Zamore 2007). siRNAs are produced from long dsRNAs MATERIALS AND METHODS of diverse origins, including the transcripts of long inverted repeats, the products of convergent transcrip- C. reinhardtii strains, culture conditions, and generation of transgenic strains: The CC-124 and Mut-11 strains have tion or RDR activity, viral RNAs, or dsRNA experimentally been previously described (Harris 1989; Zhang et al. 2002). introduced into cells (Baulcombe 2004;