Diversification of the Core RNA Interference Machinery In

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-independent 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; Chapman and CC-3491 was obtained from the Chlamy Center (http://www. Carrington 2007; Matranga and Zamore 2007). On chlamy.org). C. reinhardtii cells were routinely grown in Tris– the basis of their origins and functions, siRNAs can be acetate–phosphate (TAP) medium (Harris 1989) under mod- eong further classified into several subclasses such as primary erate light conditions at 21° (J et al. 2002). To suppress DCL1 expression by RNAi, cells were transformed by the glass- siRNAs, secondary siRNAs, heterochromatic siRNAs, beads procedure (Kindle 1990) with an inverted repeat trans- trans-acting siRNAs, and natural antisense transcript- gene designed to produce dsRNA homologous to the DCL1 derived siRNAs. These siRNAs play various roles in post- mRNA. An 360-bp fragment corresponding to part of the transcriptional regulation of gene expression, suppres- DCL1 coding sequence was amplified by reverse transcriptase– sion of viruses and transposable elements, and hetero- PCR (RT–PCR) with primers Dcr-3 (59-GCTGGAGACCCT rodersen oinnet GGGTGA-39) and Dcr-2 (59-CTGCGCGTCATTGCTGTT-39). chromatin formation (B and V 2006; This segment was then inserted in sense and antisense orienta- Vaucheret 2006; Chapman and Carrington 2007; tions, flanking a spacer sequence, in the Maa7/X IR vector Ding and Voinnet 2007; Matranga and Zamore 2007). (Rohr et al. 2004). RNAi-positive transgenic strains were identi- RNAi and Transposon Silencing 71

fied as previously described (Rohr et al. 2004). Chlamydomo- 07-030), or H3K4me3 (Abcam, ab8580). A modification- nas cells transformed with the empty Maa7/X IR vector, as a insensitive anti-H3 antibody (Abcam, ab1791) was used to adjust control, showed no effect on TOC1 or GULLIVER reactivation sample loading. (van Dijk et al. 2005 and data not shown). Chromatin immunoprecipitation assays: The H3K4 meth- DNA sequence and phylogenetic analyses: The coding ylation status of the chromatin associated with the TOC1 long sequence of the predicted DCL1 gene was confirmed by se- terminal repeat (LTR) was examined by chromatin immuno- quencing a truncated cDNA and products generated by precipitation (ChIP), following a previously described pro- reverse transcriptase–PCR amplification from polyadenylated tocol (van Dijk et al. 2005; Casas-Mollano et al. 2007). Since RNA. Polypeptides homologous to AGO-Piwi, Dicer, or RDR the antibody against H3K4me3 (Abcam) lacks specificity and were identified by BLAST or PSI-BLAST searches of protein cross-reacts with several nonhistone proteins (van Dijk et al. and/or translated genomic DNA databases. Sequences corre- 2005), this modification was not examined by ChIP.Rabbit IgG sponding to conserved Argonaute or Dicer domains were ex- (Sigma, St. Louis) was employed as a negative control. Im- tracted by comparison to the SMART database and aligned munoprecipitated DNA was quantified by real-time PCR on a with ClustalX (Thompson et al. 1997). The neighbor-joining Bio-Rad (Hercules, CA) iCycler using SYBR Green (Casas- method (Saitou and Nei 1987) and the MEGA program v3.1 Mollano et al. 2007). After each run, a melting curve was per- (Kumar et al. 2004) were used to obtain phylogenetic trees formed to ensure that no primer dimers interfered with the with Poisson-corrected amino acid distances. The coding se- quantification. The primers used for amplification of the tar- quence of DCL1 has been deposited with the EMBL/GenBank get locus have been previously reported (van Dijk et al. 2005). Data Libraries (accession no. EU368690). Cytoplasmic/nuclear fractionation of RNA: Cells from CC- DNA and RNA analyses: Standard protocols were used for 3491, which lacks a cell wall, were grown to mid-log phase in nucleic acid isolation, fractionation by gel electrophoresis, and TAP medium, collected by centrifugation, and resuspended in hybridization with 32P-labeled probes (Cerutti et al. 1997a; solution I from Keller et al. (1984). Triton X-100, instead of Sambrook and Russell 2001). To detect small RNAs by Nonidet P-40, was used to lyse the cells, and nuclei were Northern blotting, TRI reagent (Molecular Research Center) pelleted by centrifugation as previously described (Keller isolated total RNA was fractionated through Microcon YM-100 et al. 1984). The supernatant was used for the isolation of centrifugal devices (Millipore, Bedford, MA) to remove high- cytoplasmic RNA by a SDS-based protocol (Sambrook and molecular-weight transcripts. Small RNAs were concentrated Russell 2001). The pellet, consisting mostly of nuclei and cell from the filtrate by ethanol precipitation, resolved in 15% debris, was washed twice with solution I without detergent polyacrylamide/7 m urea gels and electroblotted to Hybond- (Keller et al. 1984) and then used for nuclear RNA isolation by XL membranes (GE Healthcare). Blots were then hybridized the TRI reagent procedure. Total cell RNA was purified from as previously described (Rohr et al. 2004; Ibrahim et al. 2006). CC-3491 as previously reported (Rohr et al. 2004). For analysis RT–PCR analyses: Total RNA was isolated with TRI reagent, by Northern blotting, we used cell-volume equivalent amounts according to the manufacturer’s instructions (Molecular Re- (Rudenko et al. 2003) of total (11.5 mg), cytoplasmic (10 mg), search Center), and contaminant DNA was removed by DNase- and nuclear (1.5 mg) RNAs. Small RNAs were purified from the I treatment (Ambion). First-strand cDNA synthesis and PCR cytoplasmic, nuclear, and total cell RNA fractions as described reactions were performed as previously described (Rohr et al. under DNA and RNA analyses. 2004; van Dijk et al. 2005). PCR products were resolved on 2% agarose gels and visualized by ethidium bromide staining. The number of cycles showing a linear relationship between input RESULTS RNA and the final product was determined in preliminary The core RNAi machinery in C. reinhardtii and other experiments. Controls included the use as template of reactions without RT and verification of PCR products by hybrid- green algae: We identified polypeptides related to AGO- ization with specific probes (data not shown). The primer Piwi, Dicer, or RDR by BLAST or PSI-BLAST searches of sequences were as follows: for DCL1, Dcr-7 (59-GCAGCATCG protein and/or translated genomic DNA databases. ATGGTACTGATAGC-39) and Dcr-10 (59-CTGCACGTGCTTG Since several of the examined genomes are in draft 9 9 CTTGGAT-3 ); for DCL2,Dicer2-F4(5 -CAGTGTGTGCGAGCT stage, an important caveat in our analyses is that some GGAG-39) and Dicer2-R3 (59-GGACATGGCCTCGGCACT-39); for DCL3,Dicer3-F3(59-GTGGTGTCCTTCTGGCTGTTC-39) proteins may be missing from the databases whereas and Dicer3-R3 (59-GTTGCTGACCAGCGCCTTG-39); and for others may have errors in the predicted gene structure. ACT1, ACT-cod-F (59-GACATCCGCAAGGACCTCTAC-39)and However, we considered as potential homologs only ACT-cod-R (59-GATCCACATTTGCTGGAAGGT-39). proteins that exhibited enough sequence similarity to be Nuclear run-on transcription assays: Chlamydomonas cells aligned together and to be used for phylogenetic tree grown to mid-log phase were concentrated by centrifugation, resuspended to a density of 1 3 108 cells/ml in wash buffer construction. The core RNAi machinery components (10 mm Tris–HCl, pH 8.0, 0.5 mm EDTA, 50 mm KCl, 250 mm seem to be entirely absent from algal species with small sucrose, and 1 mm DTT), and frozen in liquid nitrogen. These nuclear genomes such as the red alga Cyanidioschyzon permeabilized cells were then used to determine transcrip- merolae (an 16.5-Mb nuclear genome) and the green eller tional activity as previously described for isolated nuclei (K algae Ostreococcus lucimarinus and Ostreococcus tauri (an et al. 1984; Cerutti et al. 1997a). Nuclear transcripts, labeled with ½32PrUTP,were employed as probes on filters containing an 12.6-Mb nuclear genome) (data not shown). This is excess of target DNA. The radioactivity retained on the mem- consistent with the proposal that the RNAi mechanism branes was quantified with a PhosphorImager (GE Healthcare) appears to have been lost independently several times (Cerutti et al.1997a,b). during eukaryotic evolution (Cerutti and Casas- Immunoblot analyses: Histone methylation status was ex- Mollano 2006; Nakayashiki et al. 2006). In contrast, amined in vivo by Western blotting, as previously described (van Dijk et al.2005;Casas-Mollano et al. 2007). The different obvious AGO and Dicer-like homologs are present in methylated states of histone H3 Lys 4 were detected with anti- Chlamydomonas, the related volvocine alga Volvox carteri bodies against H3K4me1 (Abcam, ab8895), H3K4me2 (Upstate, f. nagariensis as well as in another green alga, Chlorella sp. 72 J. A. Casas-Mollano et al.

Figure 1.—Neighbor-joining tree showing the phylogenetic relationship among Argonaute proteins. Sequences corresponding to the PAZ and Piwi domains of each polypeptide were aligned using the ClustalX program and the tree was drawn using the MEGA v3.1 program. Numbers indicate bootstrap values, .60%, based on 1000 pseudoreplicates. Polypeptides belonging to the green alga lineage are shaded. Species are designated by a two-letter abbreviation preceding the name of each protein: At, A. thaliana;Cr,C. reinhardtii; Cs, Chlorella sp. NC64A; Dm, D. melanogaster; Hs, H. sapiens; Os, O. sativa; and Vc, V. carteri f. nagariensis. Accession numbers of proteins used to draw the tree are the following: At AGO1, AAC18440; At AGO2, NP_174413; At AGO3, NP_174414; At AGO4, NP_565633; At AGO5, NP_850110; At AGO6, NP_180853; At AGO7(ZIP), NP_177103; At AGO8, NP_197602; At AGO9, NP_197613; At AGO10(PNH), CAA11429; Cr AGO1, XP_001694840; Cr AGO2, XP_001698670; Cr AGO3, XP_001698906; Dm Ago1, BAA88078; Dm Ago2, Q9VUQ5; Cs NC64A scaffold-12-45 gene25, v2_NC64A_scaffold_12_45_gene25; Hs AGO1(eIF2C-1), AAH63275; Hs AGO2(eIF2C-2), AAL76093; Hs AGO3(eIF2C-3), BAB14262; Hs AGO4(eIF2C-4), BAB13393; Os AGO701, XP_468547; Os AGO702, BAB96813; Os AGO703, NP_912975; Os AGO704, XP_478040; Os AGO705, AL606693; Os AGO706, NP_909924; Os AGO707, AP005750; Os AGO708, XP_473529; Os AGO709, XP_473887; Os AGO710, XP_469312; Os AGO711, AP004188; Os AGO712, XP_476934; Os AGO713, XP_473888; Os AGO714, XP_468898; Os AGO715, XP_477327; Os AGO716, XP_464271; Os AGO717, XP_469311; Os AGO719, AP000836; Vc_105714, v1_105714; Vc_92637, v1_92637. Accession numbers in italics are according to the annotated draft genomes of V. carteri (http://genome.jgi-psf.org/Volca1/Volca1.home.html) and Chlorella sp. NC64A (http://greengene.uml. edu/chlorella/chlorella.html).

NC64A, an endosymbiont of the ciliated protozoan polypeptides in the genome of the latter species, which, Paramecium bursaria (Figures 1 and 2). As in the case of considering their existence in Chlorella sp. NC64A, land plants (Cerutti and Casas-Mollano 2006; Aravin suggests that the draft genome of C. vulgaris C-169 is et al. 2007), algae appear to lack Piwi proteins. most likely incomplete. This issue may also apply to The dual domain structure of the AGO-Piwi polypep- the other algal species. In Arabidopsis, the biogenesis tides, namely a PAZ domain followed by a Piwi domain of phased trans-acting siRNAs requires RDR activity (Figure 3), has been well conserved in the three species (Allen et al. 2005; Yoshikawa et al. 2005; Chapman of algae, although some of the predicted AGO gene and Carrington 2007). Chlamydomonas also contains models may be partly incorrect or incomplete due to phased siRNAs (Molnar et al. 2007; Zhao et al. 2007), gaps in the available genomic sequences (data not despite an apparent lack of an RDR. However, a long shown). The Dicer enzymes initially characterized in dsRNA generated by convergent transcription and D. melanogaster and humans are multidomain proteins annealing of the complementary RNAs or a foldback consisting of RNA helicase domains, a motif of un- transcript of long inverted repeats could also be cleaved known function (DUF283) that appears to be a di- by Dicer in a processive fashion to generate phased, vergent dsRNA-binding domain (dsRB) (Dlakic 2006), RDR-independent siRNAs. Given these uncertainties, a PAZ domain, two RNaseIII catalytic domains, and the question of whether C. reinhardtii encodes an RDR a dsRNA-binding domain (Bernstein et al. 2001; homolog will likely remain open until all gaps in the Meister and Tuschl 2004; Cerutti and Casas-Mol- current version of the genome are ﬁlled. lano 2006; Margis et al. 2006). This overall organiza- To gain insight into the evolution and diversity of the tion is maintained in Dicer-like proteins from the RNA-mediated silencing machinery among algae, phy- volvocine algae except for the absence of obvious PAZ logenetic analysis of the Argonaute and Dicer-like pro- and dsRB domains (Figure 3; Zhao et al. 2007). In teins was performed with the available algal sequences contrast, an RDR was not identiﬁed in the genomes of as well as with those from two land plants (A. thaliana Chlamydomonas, Volvox, or Chlorella sp. NC64A, but a and Oryza sativa) and two metazoans (D. melanogaster clear homolog was predicted from the sequence of the and Homo sapiens). In C. reinhardtii, three paralogs of free-living Chlorella vulgaris C-169 (data not shown). AGO (AGO1, AGO2, and AGO3) and of Dicer-like Interestingly, we could not detect AGO or Dicer-related (DCL1, DCL2, and DCL3) proteins have been identi- RNAi and Transposon Silencing 73

polypeptide whereas Chlorella sp. NC64A has only one copy of each protein in the respective draft versions of their genomes (Figures 1 and 2). The examined Argo- naute and Dicer-like polypeptides fell into three relatively well-supported groups that included exclusively proteins from land plants, algae, or animals (Figures 1 and 2). The only exception was D. melanogaster Ago2 that clustered with the algal Argonaute polypeptides (Figure 1), but this is likely a methodological artifact resulting from the unusually fast evolution of the fly gene (see below). AGO proteins have undergone a marked degree of expansion in most of the eukaryotic lineages analyzed (Figure 1), conceivably associated with extensive functional diversification. In land plants, several duplica- tions of AGO polypeptides appear to have occurred igure both before and after the divergence of dicots and mono- F 2.—Phylogenetic tree of Dicer-like proteins. Se- cots, represented by A. thaliana and O. sativa, respec- quences corresponding to the RNaseIII domains were aligned using the ClustalX program, and a neighbor-joining tree was tively (Figure 1). Expansion of Argonaute proteins has constructed using MEGA v3.1. Numbers show bootstrap val- also occurred in the algal lineages, particularly in C. ues, .60%, based on 1000 pseudoreplicates. Polypeptides be- reinhardtii (Figure 1). However, our phylogenetic anal- longing to the green alga lineage are shaded. Species are ysis indicates that gene duplication and, likely, pathway designated by a two-letter abbreviation preceding the name diversification occurred after the divergence of green of each protein, as described in the legend to Figure 1. Acces- sion numbers of proteins used to draw the tree are the follow- algae from the lineage leading to land plants. A phylo- ing: At DCL1, NP_171612; At DCL2, NP_566199; At DCL3, genetic tree of Dicer-like proteins supports a similar NP_189978; At DCL4, NP_197532; Cr DCL1, EU368690; Cr conclusion (Figure 2). DCL2, XP_001698921; Cr DCL3, XP_001692436; Cs NC64A Most animals appear to encode a single Dicer gene, scaffold-5-57 gene7, v2_NC64A_scaffold_5_57_gene7;Dm with the exception of insects that contain two. Whereas Dcr1, Q9VCU9; Dm Dcr2, BAB69959; Hs DCR1, NP_803187; Os DCL1, NP_912466; Os DCL2a, XP_463068; insect Dcr1 clusters with all other animal Dicers, Dcr2 is Os DCL2b, BAD34005; Os DCL3a, XP_463595; Os DCL3b, much more divergent and forms a paralogous clade NP_922059; Os DCL4, XP_473129; Vc_106340, v1_106340.Ac- (Cerutti and Casas-Mollano 2006). Insect Ago2 is cession numbers in italic are according to the annotated draft also much more divergent than Ago1 and does not genomes of V. carteri (http://genome.jgi-psf.org/Volca1/ cluster with most other animal Argonaute-like proteins Volca1.home.html) and Chlorella sp. NC64A (http://greengene. erutti asas ollano uml.edu/chlorella/chlorella.html). (Figure 1; C and C -M 2006). In D. melanogaster, Dcr1 and Ago1 are involved in the miRNA pathway whereas the divergent Dcr2 and Ago2 play a fied (Figures 1 and 2). Moreover, 23-nt transgenic role in siRNA-mediated processes (Okamura et al. 2004; siRNAs (Rohr et al. 2004; Ibrahim et al. 2006) and Zamore and Haley 2005). Moreover, Dcr2 and Ago2 21-nt transposon small RNAs (van Dijk et al. 2006) have been implicated in antiviral immune responses have been detected by Northern blotting, suggesting (Zambon et al. 2006; Ding and Voinnet 2007) and a that, in Chlamydomonas, different Dicers may be func- recent report suggests that both Dcr2 and Ago2 are tional and generate small RNAs of various lengths. Volvox among the fastest-evolving genes in flies, perhaps as carteri contains two AGO paralogs and one Dicer-like a result of a co-evolutionary ‘‘arms race’’ with viral

Figure 3.—Genomic organization of C. reinhardtii DCL1 and AGO1 and sche- matic of the corresponding proteins. (Top) The chromosomal arrangement of the DCL1 and AGO1 genes, which are transcribed in divergent orientation (arrows) on linkage group II (http:// genome.jgi-psf.org/Chlre3/Chlre3.home. html). Polyadenylation sites are indicated by the stop signs. (Middle) The precursor messenger RNAs (excluding 59 and 39 untranslated regions) with exons indicated by solid boxes. The annealing sites of primers used for RT–PCR amplification are shown below the exons. (Bottom) The domain architecture of the DCL1 and AGO1 proteins. Black oval, nuclear localization signal; PAZ, Piwi/Argonaute/Zwille domain; Piwi, Piwi domain; DEXHc, DEAD/DEAH-like helicase superfamily domain; HelC, helicase superfamily C-terminal domain; DUF283, putative divergent dsRNA-binding fold; RIII, ribonucleaseIII C-terminal catalytic domain. 74 J. A. Casas-Mollano et al. pathogens (Obbard et al. 2006). In Arabidopsis, DCL2, without altering DCL2 or DCL3 mRNA levels ½Figure 4A, DCL3, and DCL4, which function in a variety of siRNA- CC-124(Dcl1-IR); data not shown. mediated silencing pathways (including virus defense Long antisense RNAs corresponding to the TOC1 and transposon repression) (Brodersen and Voinnet retrotransposon have been previously observed in 2006; Vaucheret 2006; Chapman and Carrington certain Chlamydomonas strains and postulated to arise 2007; Ding and Voinnet 2007), are also more divergent by readthrough transcription from a promoter(s) flank- than DCL1, which generates miRNAs (Bartel 2004; ing a specific transposon copy (Day and Rochaix Margis et al. 2006). Interestingly, C. reinhardtii DCL1 1991). Moreover, by genetic tetrad analyses, the pro- and AGO1 do not cluster tightly with their counterparts duction of sense and antisense transcripts in the same in the closely related alga V. carteri and appear to be cell, presumably annealing into dsRNA that triggers more divergent than the other paralogs (Figures 1 RNAi, has been shown to inhibit TOC1 RNA accumula- and 2). tion (Day and Rochaix 1991). In agreement with C. reinhardtii DCL1 and AGO1 are encoded by ad- these findings, we have detected siRNAs derived from jacent, divergently transcribed genes: By analogy to the TOC1 LTRs in CC-124 (Figure 4B), although they insects and land plants, we hypothesized that Chlamy- appear to be present at very low levels. This observation domonas DCL1 and AGO1, which seem to be more is consistent with the small number of transposon divergent than the other paralogs, may be involved in siRNAs identified by high-throughput sequencing in defense responses against genomic parasites such as Chlamydomonas (Molnar et al. 2007; Zhao et al. viruses and transposable elements. Therefore, we de- 2007).Inaddition,wewereunabletodetectGULLIVER cided to examine their genes in more detail. The amino siRNAs by Northern blotting in the wild-type background acid sequence of DCL1 has been previously inferred (Figure 4B). from genomic DNA sequences by computational pre- RNAi-mediated suppression of DCL1 did reduce the diction of exon/intron regions. To obtain more accu- steady-state level of TOC1 siRNAs (Figure 4B). However, rate information on this protein, a truncated cDNA and this resulted in only a very slight increase in TOC1 RT–PCR products were used to determine the actual transcripts in comparison with the wild type (Figure coding sequence and exon/intron boundaries (Figure 4C). Furthermore, the TOC1 transposition frequency 3). The coding region of AGO1 was not verified ex- did not appear to be affected. Southern blot analyses of perimentally but both the 59- and the 39-ends of the 10 parallel subcultures of CC-124 and CC-124(Dcl1-IR) predicted model are supported by available ESTs. In- revealed no additional TOC1 copies in the strain where terestingly, the genes encoding DCL1 and AGO1 are DCL1 expression was downregulated (Figure 4D and arranged head to head near the distal end of linkage data not shown). GULLIVER transposition was also group II. Their respective translation start sites are unchanged by RNAi-mediated DCL1 suppression (data separated by only 300 bp (Figure 3), suggesting that not shown). Interestingly, the full-length, transposition these genes may be transcriptionally coregulated by ex- competent TOC1 RNA, which is 5.5 kb (Day et al. 1988; pression from a common bidirectional promoter and, Day and Rochaix 1991), was virtually undetectable in conceivably, part of the same RNAi pathway. Northern blots of the CC-124 and CC-124(Dcl1-IR) DCL1 appears to play a minor role in transposon strains (Figure 4C). The majority of the observed silencing in wild-type C. reinhardtii grown under TOC1 transcripts were ,5.5 kb and very heterogeneous standard laboratory conditions: Several partly active in size, giving rise to a smeary signal on the RNA blots transposable elements have been described in Chlamy- (Figure 4C). Indeed, on the basis of the available domonas, including two class I retrotransposons, TOC1 Chlamydomonas ESTs, most TOC1 transcripts in wild- and REM1, and a number of class II DNA elements, type cells appear to be prematurely terminated RNAs, GULLIVER, PIONEER, TCR1, TCR2, and TCR3 (Day solo LTRs, or transcripts initiated from flanking pro- et al. 1988; Ferris 1989; Schnell and Lefebvre 1993; moters that extend and terminate into a TOC1 element Wang et al. 1998; Pe´rez-Alegre et al. 2005). To test (data not shown). directly the role of DCL1 in defense responses against DCL1 is required for the post-transcriptional repres- some of these mobile genetic elements, we decided to sion of the TOC1 retrotransposon in a mutant strain suppress its expression by RNAi. The wild-type Chlamy- defective in transcriptional silencing: We reasoned that domonas strain CC-124 was transformed with an in- the very modest effect of DCL1 suppression on TOC1 verted repeat construct designed to produce dsRNA reactivation in wild-type Chlamydomonas might be due homologous to DCL1 (Dcl1-IR). However, the DNA to the fact that this retrotransposon (in particular its sequence chosen to build the inverted repeat is quite full-length, transposition-competent form) is silenced divergent from the corresponding regions in DCL2 or mainly at the transcriptional level by a DCL1-independent DCL3, to avoid the unintended downregulation of mechanism(s). Indeed, we have previously demon- expression of the latter genes. By using this approach, strated that both TOC1 and GULLIVER are repressed we recovered several transformants displaying DCL1 sup- by effectors that operate at the transcriptional level pression (to 25% of the wild-type transcript amounts) (Jeong et al. 2002; Zhang et al. 2002). MUT11 encodes a RNAi and Transposon Silencing 75

Figure 4.—RNAi-mediated suppression of DCL1 affects the post-transcriptional silencing of the TOC1 retrotransposon. (A) RT– PCR analysis of DCL1, DCL2, and DCL3 expression in the indicated strains. Reactions using RNA not treated with reverse transcriptase (RT) as the template were employed as a negative control. Amplification of ACT1 (encoding actin) transcripts is shown as an input control. Numbers below the panels indicate relative levels of specific transcripts normalized to the ACT1 mRNA amount. CC-124, wild-type C. reinhardtii; CC-124(Dcl1-IR), CC-124 transformed with an inverted repeat (IR) transgene designed to produce dsRNA homologous to DCL1; Mut-11, mutant defective in a core subunit of H3K4 methyltransferase complexes; Mut- 11(Dcl1-IR), Mut-11 transformed with an IR transgene designed to induce RNAi of DCL1. (B) Northern blot detection of transposon siRNAs. Column-fractionated small RNAs were separated in a 15% denaturing polyacrylamide gel, electroblotted onto a nylon membrane, and hybridized with a probe corresponding to the right terminus of the GULLIVER transposon (middle). The same blot was then sequentially reprobed for the TOC1 LTR (top) and for the U6 small nuclear RNA (bottom) as a loading control. (C) Northern blot of total cell RNA probed sequentially for TOC1 (top) to examine transcript levels and for ACT1 (bottom) to test for equivalent loading of the lanes. The asterisk indicates the full-length, 5.5-kb TOC1 transcript. (D) Southern blot analysis of TOC1 transposition. Genomic DNA from parallel subcultures (clones) of the indicated strains was digested with HincII and probed for TOC1. The asterisks indicate newly integrated transposon copies in the subclones of Mut-11(Dcl1-IR). subunit of histone methyltransferase complexes and its GULLIVER siRNAs (Figure 4B), which correlated with deficiency results in the loss of histone H3 lysine 4 a pronounced increase in TOC1 transcripts (Figure monomethylation (H3K4me1), enrichment in H3K4 4C). Moreover, an 5.5-kb, presumably transposition- dimethylation (H3K4me2), a decrease in H3K4 trime- competent, TOC1 RNA was clearly detectable in Northern thylation (H3K4me3), and the reactivation of silenced blots of the Mut-11(Dcl1-IR) strain (Figure 4C). The transgenes and transposons ( Jeong et al. 2002; van Dijk occurrence of TOC1 transposition was accordingly et al. 2005). In nuclear run-on assays, TOC1 transcription enhanced in Mut-11(Dcl1-IR). Parallel subcultures of was significantly enhanced in the Mut-11 mutant whereas the transgenic strain showed additional TOC1 copies DCL1 suppression in an otherwise wild-type background integrated into the genome at a much higher frequency had no effect ½Figure 5A, cf. CC-124(Dcl1-IR) and Mut-11. than those from Mut-11 (Figure 4D and data not shown), We also observed a substantial increase in transposon- although the latter strain also accumulated, over a longer derived siRNAs (both TOC1 and GULLIVER small time scale, extra TOC1 insertions relative to the wild-type RNAs) in Mut-11 (Figure 4B). We speculate that an en- CC-124. However, by nuclear run-on assays, DCL1 sup- hancement in overall genome transcription in Mut-11, pression in the Mut-11 mutant background did not en- due to defects in chromatin repression, results in greater hance global TOC1 transcription ½Figure 5A: cf. Mut-11 production of both sense and antisense transposon tran- and Mut-11(Dcl1-IR). scripts that likely anneal, producing increased amounts We also examined whether downregulation of DCL1 of dsRNAs processed by Dicer into siRNAs. expression had any effect on DNA or chromatin To further explore the connection between DCL1 modifications associated with the silenced transpos- and transposon silencing, we also suppressed DCL1 ex- able elements. Cytosine DNA methylation was analyzed pression by transgenic RNAi in the Mut-11 mutant back- by digestion with methylation-sensitive isoschizomers. ground ½Figure 4A, Mut-11(Dcl1-IR); data not shown. HpaII and MspIrecognizethesameDNAsequence This resulted in a decrease in the levels of TOC1 and (59-CCGG-39), but HpaII is inhibited by methylation of 76 J. A. Casas-Mollano et al.

Figure 5.—DCL1 is dispensable for transcriptional silencing of the TOC1 retrotransposon. (A) Tran- scriptional activity of TOC1 in nuclear run-on assays of the indicated strains, described in the legend to Figure 4. Transcription of TUBA (encoding a-tubulin) and ACT1 (encoding actin) was evaluated as a control. (B) Southern blot analysis of TOC1 and GULLIVER cytosine DNA methylation with isoschizomeric restric- tion enzymes (REs). Total cell DNA was digested with the indicated REs, resolved by agarose gel electrophoresis, and probed with the TOC1 LTR (left) or with the right terminus of GULLIVER (right). In the absence of cytosine methylation, HinfI/MspI or HinfI/HpaII cleavage would result in a TOC1 fragment of 140 bp, whereas XbaI/MspIorXbaI/HpaII cleavage would result in a GULLIVER fragment of 210 bp. The multiple segments obtained with HinfI or XbaI digestions reflect sequence hetero- geneity among different copies of TOC1 or GULLIVER. (C) Immuno- blot analysis of in vivo H3K4 methylation states. Whole-cell protein extracts from the indicated strains were separated by SDS–PAGE, transferred to nitrocellulose, and probed with antibodies raised against mono-, di-, or trimethyl H3K4. Sample loading was calibrated on the basis of immunoblots with a modification-insensitive anti-H3 antibody. (D) ChIP assay of H3K4 modifications associated with the TOC1 LTR. ChIP was performed with anti-H3K4me1, anti-H3K4me2, or anti- H3 antibodies. A rabbit IgG antibody was used as a negative control. Immunoprecipitated DNA was examined by real-time PCR and enrichment was calculated relative to the anti-H3 immunoprecipitate. For illustration purposes, the level of H3K4me1 in the CC-124 strain and the level of H3K4me2 in the Mut-11 mutant were set to 1.0 and the remaining samples adjusted accordingly in the bar graph. Results represent the mean 6SD of three independent experiments. either cytosine, whereas MspI is sensitive only to meth- would promote gene activation whereas a reduction in ylation of the outer cytosine residue. Thus, if HpaII/ H3K4me2 would counteract transcriptional compe- MspI sites become methylated, the inability of the en- tence (van Dijk et al. 2005), and they are likely the zymes to cleave will result in the appearance of DNA result of indirect or stochastic effects. Moreover, neither fragments of higher molecular weight. By using this of these changes seemed to have any consequence for approach, we observed that both TOC1 and GULLIVER the global transcriptional activity of TOC1 (Figure 5A). showed very little, if any, cytosine DNA methylation and Thus, our results suggest that DCL1 functions mainly in that this modification was not affected by RNAi-medi- the post-transcriptional silencing of retrotransposons ated suppression of DCL1 (Figure 5B and data not such as TOC1. In contrast, it appears to have no role in shown). As previously reported (van Dijk et al. 2005), the maintenance of their transcriptional repression or, loss of MUT11 causes substantial changes in global levels alternatively, such an activity may be redundantly com- of H3K4 methylation, detected by immunoblotting of pensated by one of the other Dicer paralogs. Although whole-cell proteins with modification-specific antibod- all transcripts with homology to the TOC1 LTRs (the ies (Figure 5C). However, DCL1 suppression had virtu- sequence corresponding to the TOC1 siRNAs) appear to ally no effect on the overall levels of these histone be targeted by the DCL1-dependent mechanism ½Figure modifications (Figure 5C). We also examined directly 4C: cf. Mut-11 and Mut-11(Dcl1-IR), the key to prevent- the chromatin environment of the TOC1 retrotranspo- ing transposition is likely the suppression of the full- son by chromatin immunoprecipitation assays. In these length TOC1 RNA. experiments, we did detect somewhat reduced amounts TOC1 transcripts and siRNAs are predominantly of H3K4me1 associated with the TOC1 transcription located in the nucleus: To gain insight into the sub- units in CC-124(Dcl1-IR) and slightly lower levels of cellular localization of the DCL1-dependent mechanism H3K4me2 in Mut-11(Dcl1-IR) in comparison with the of TOC1 suppression, we examined the distribution of respective control strains (Figure 5D). However, these TOC1 long RNAs as well as siRNAs between the nucleus are conflicting outcomes, since a decrease in H3K4me1 and the cytoplasm. For these experiments, we used a RNAi and Transposon Silencing 77

ization signal (Figure 3). Thus, it seems likely that the DCL1-dependent pathway of post-transcriptional retrotransposon silencing operates partly, if not entirely, in the nucleus of C. reinhardtii.

DISCUSSION In the past few years, RNA-mediated silencing has emerged as a widespread mechanism to control gene expression in eukaryotes. Moreover, in land plants, animals, and filamentous fungi, the core RNAi machinery has significantly expanded and diversified (Carmell Figure 6.—Subcellular compartmentalization of TOC1 et al. 2002; Cerutti and Casas-Mollano 2006; Margis transcripts and siRNAs. (A) Northern blot analysis of TOC1 et al. 2006; Nakayashiki et al. 2006). In these organisms, long RNAs in whole cells (WHOLE), the cytoplasmic fraction (CYT), or the nuclear fraction (NUC) from a cell-wall-less various small RNA-silencing pathways often involve strain (CC-3491). Purified RNAs were resolved on agarose– different sets of Dicer, AGO-Piwi, and/or RDR (when formaldehyde gels and hybridized to a TOC1 probe. The same present) proteins, although there is also a consider- blot was reprobed for ACT1, a predominantly cytoplasmic able degree of operative overlap (Okamura et al. 2004; transcript, and for the U6 small nuclear RNA, retained in Brodersen and Voinnet 2006; Nakayashiki et al. the nucleus, to assess the effectiveness of the cell fraction- aucheret hapman arrington ation. (B) Northern blot detection of TOC1 siRNAs in the dif- 2006; V 2006; C and C ferent subcellular fractions. Column-purified small RNAs 2007). This functional diversification is just beginning were separated in a 15% denaturing polyacrylamide gel, elec- to be elucidated, but the presence of duplicated RNAi troblotted onto a nylon membrane, and sequentially hybrid- components appears to allow the evolution of new gene ized with probes corresponding to the TOC1 LTR and the U6 control mechanisms that use small RNAs as sequence- small nuclear RNA. specific determinants, as well as more effective strategies to counteract the action of invading genomic parasites C. reinhardtii strain lacking the cell wall (CC-3491), such as viruses and transposable elements (Brodersen which facilitated the isolation of intact nuclei (Keller and Voinnet 2006; Cerutti and Casas-Mollano 2006; et al. 1984). Day and Rochaix (1991) have previously Vaucheret 2006; Chapman and Carrington 2007; reported that TOC1 transcripts are mainly nonpolyade- Matranga and Zamore 2007). In contrast, many uni- nylated, suggesting that they may be retained in the cellular eukaryotes, particularly those with small nuclear nucleus. Our cell fractionation analyses indicated that genomes, seem to have lost entirely the RNAi machinery the majority of the long TOC1 transcripts are present in or have retained only a basic set of RNAi components the nucleus (Figure 6A), although an abundant RNA of (Cerutti and Casas-Mollano 2006; Nakayashiki et al. 2.7 kb appears to be mostly cytoplasmic. This partic- 2006). Indeed, it has been proposed that the deploy- ular RNA is shorter than the described full-length TOC1 ment of small RNA pathways for spatiotemporal regu- element (Day et al. 1988) and was not prevalent in the lation of the transcriptome has shaped the evolution of CC-124 strain (Figure 4C), indicating that it may have eukaryotic genomes and contributed to the complexity originated from a newly inserted TOC1 element in CC- of multicellular organisms (Chapman and Carrington 3491. Interestingly, the TOC1 small RNAs were also 2007). found predominantly in the nuclear fraction (Figure Our results indicate that the unicellular eukaryote C. 6B). The localization of the full-length 5.5-kb TOC1 reinhardtii has also undergone extensive duplication of RNA could not be assessed because, as is the case for Argonaute and Dicer polypeptides after the divergence CC-124, it was undetectable in Northern blots of the of the green algae and land plant lineages. Moreover, CC-3491 strain (Figure 6A). Chlamydomonas contains an assortment of siRNAs as As controls in our compartmentalization analyses, we well as miRNAs (Molnar et al. 2007; Zhao et al. 2007). examined the distribution of the actin mRNA, which is Other unicellular organisms with relatively large nu- mainly a cytoplasmic transcript (Rudenko et al. 2003), clear genomes, such as the ciliate Tetrahymena thermo- and the U6 small nuclear RNA, which is thought to phila and the social amoeba Dictyostelium discoideum, remain in the nucleus (Stanek and Neugebauer 2006) also show expansion of the core RNAi machinery and (Figure 6, A and B). Our findings indicate that both different classes of small RNAs (Lee and Collins 2006; the long TOC1 transcripts, which are the targets of Hinas et al. 2007). These observations suggest that post-transcriptional degradation by a DCL1-dependent diversification of RNA-silencing components is not mechanism (Figure 4C), and the TOC1 small RNAs, limited to multicellular lineages and appears to have which appear to be generated by DCL1 (Figure 4B), are been a common occurrence during the evolution of mainly in the nucleus. Moreover, the DCL1 (as well as eukaryotes. However, it might have provided a selec- the AGO1) protein contains a predicted nuclear local- tive advantage, resulting in its genetic fixation only in 78 J. A. Casas-Mollano et al. organisms with relatively complex genomes and life addition, transcriptional silencing mediated by the cycles (regardless of cellularity). MUT11 machinery and involving H3K4 monomethyla- Despite considerable advances in our mechanistic tion seems to be independent of RNAi. We have never understanding of RNA-mediated silencing, the biolog- detected small RNAs corresponding to single-copy ical functions of different RNAi pathways remain largely transgenes silenced by this mechanism either in wild- uncharacterized in most eukaryotes. To begin address- type or in mutant strains, and DCL1 suppression had no ing this issue in C. reinhardtii, we focused on the role(s) effect on the transcriptional repression of, or on the of DCL1. This protein and AGO1 are more divergent chromatin modifications associated with, TOC1 or an than the other paralogs encoded in the Chlamydomo- RbcS2:aadA:RbcS2 transgene (this work and data not nas genome, suggesting, by analogy to D. melanogaster shown). However, we cannot exclude a redundant Dcr2 and Ago2 (Obbard et al. 2006), that they may be involvement of DCL2 and/or DCL3 in the maintenance involved in siRNA-mediated defense responses against of transcriptional silencing in Chlamydomonas. genomic intruders. The head-to-head chromosomal In higher plants, transposons appear to be regu- arrangement of their genes also indicated that they lated by a variety of epigenetic mechanisms (Feschotte might be coregulated and, possibly, part of the same et al. 2002; Lippman et al. 2003; Rudenko et al. 2003; pathway. However, RNAi-mediated suppression of DCL1 Lippman and Martienssen 2004; Woodhouse et al. had virtually no effect on the reactivation of the 2006; Rangwala and Richards 2007; Slotkin and retrotransposon TOC1 or the DNA element GULLIVER Martienssen 2007). In Arabidopsis, the analysis of a in a wild-type Chlamydomonas strain. In contrast, when number of mutants revealed that DNA methylation, TOC1 transcription was elevated, due to a defect in post-translational histone modifications as well as chro- chromatin-mediated silencing in Mut-11 (Zhang et al. matin packaging and condensation contribute to trans- 2002; van Dijk et al. 2005), DCL1 played a substantial poson silencing, although to different extents, depending role in the post-transcriptional suppression of this on the specific transposable element (Lippman et al. retrotransposon. Interestingly, the deficiency in post- 2003; Rangwala and Richards 2007; Slotkin and transcriptional silencing caused by DCL1 suppression Martienssen 2007). RNAi seems to play a role in the was not compensated by DCL2 or DCL3. Thus, our sequence-specific targeting of transposons and other findings suggest that DCL1 (and presumably AGO1) repeated sequences for heterochromatin formation may be part of an RNAi pathway that has specialized for (Woodhouse et al. 2006). Many endogenous siRNAs the control of transposable elements in Chlamydomo- in Arabidopsis correspond to silenced transposable nas. Moreover, based on the subcellular distribution of elements, and an RDR (RDR2), a Dicer homolog TOC1 transcripts and siRNAs, this mechanism most (DCL3), and an Argonaute (AGO4) are required for the likely operates (at least partly) in the nucleus. To our production of transposon-derived siRNAs (Lippman knowledge, viruses naturally infecting C. reinhardtii have and Martienssen 2004; Xie et al. 2004; Matzke et al. not been isolated and, therefore, it remains to be 2007; Slotkin and Martienssen 2007). However, most explored whether DCL1 could also be involved in repressed transposons are not immediately reactivated antiviral immunity. In addition, DCL1 appears to have in mutants of the RNAi machinery (Lippman et al. 2003; no effect on miRNA processing since the levels of several Zilberman et al. 2003; Woodhouse et al. 2006), even endogenous miRNAs were not affected in the DCL1- when siRNAs are completely lost (Xie et al. 2004). This suppressed strains (data not shown), although we can- suggested that RNAi may be involved in the establish- not rule out a redundant, overlapping function of DCL2 ment of transcriptional transposon silencing, as dem- and/or DCL3. onstrated for FWA transgenes in Arabidopsis (Chan The TOC1 and GULLIVER transposons are present as et al. 2004), but perpetuation of their repression can dispersed repeats in the C. reinhardtii genome at 10–40 occur by alternative means, such as maintenance DNA copies/haploid genome and appear to integrate pref- methylation (Lippman and Martienssen 2004; Tran erentially in euchromatic regions (Day et al. 1988; et al. 2005; Matzke et al. 2007; Slotkin and Martienssen Ferris 1989; Hall and Luck 1995; Kim et al. 2006; 2007). Yet a redundant role of RNAi components in Merchant et al. 2007). In fact, some of these transpos- the maintenance of transcriptional silencing and/or able elements were first isolated due to the mutant the existence of transcriptional repression mecha- phenotypes caused by their insertion into active genes nisms entirely independent of RNAi is also possible (Day et al. 1988; Schnell and Lefebvre 1993). Both (Woodhouse et al. 2006; Rangwala and Richards TOC1 and GULLIVER seem to be controlled primarily at 2007; Slotkin and Martienssen 2007). In addition, a the transcriptional level, when cells are grown under function of the RNAi machinery in the post-transcrip- standard laboratory conditions (this work and Jeong tional silencing of transposons remains to be examined et al. 2002). However, their DNA does not appear to be in higher plants. heavily methylated, suggesting that, in Chlamydomo- In Neurospora crassa, the RNAi machinery is not nas, transposon siRNAs do not direct methylation of required for heterochromatin formation, H3K9 meth- (presumably trans-) complementary DNA sequences. In ylation, or DNA methylation (Chicas et al. 2004; Freitag RNAi and Transposon Silencing 79 et al. 2004), suggesting that it has no role in transcrip- Baulcombe, D., 2004 RNA silencing in plants. Nature 431: 356–363. aumberger aulcombe tional silencing. However, it controls the expression of B ,N.,andD.C.B , 2005 Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits micro- transposon and other repetitive sequences post-tran- RNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA scriptionally by directing the degradation of cognate 102: 11928–11933. ernstein audy ammond annon transcripts (Chicas et al. 2004; Nolan et al. 2005). In C. B , E., A. A. C ,S.M.H and G. J. H , 2001 Role for a bidentate ribonuclease in the initiation step elegans, silencing of the Tc1 DNA transposon also ap- of RNA interference. Nature 409: 363–366. pears to be (at least in part) post-transcriptional (Sijen Bestor, T. H., 2003 Cytosine methylation mediates sexual conflict. and Plasterk 2003). Likewise, our findings in the green Trends Genet. 19: 185–190. Brodersen, P., and O. Voinnet, 2006 The diversity of RNA silenc- alga Chlamydomonas indicate that the TOC1 retrotrans- ing pathways in plants. Trends Genet. 22: 268–280. poson can be suppressed post-transcriptionally by RNA Carmell,M.A.,Z.Xuan,M.Q.Zhang and G. J. Hannon, interference. Yet this alga also relies on a DCL1- 2002 The Argonaute family: tentacles that reach into RNAi, de- velopmental control, stem cell maintenance, and tumorigenesis. independent, transcriptional silencing mechanism(s) Genes Dev. 16: 2733–2742. to control mobile genetic elements. Interestingly, chro- Casas-Mollano, J. A., K. van Dijk,J.Eisenhart and H. Cerutti, matin-mediated repression involving the MUT11 ma- 2007 SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chla- chinery is sensitive to temperature, being much more mydomonas. Nucleic Acids Res. 35: 939–950. effective at 17° than at 25° (Cerutti et al. 1997b). Con- Cerutti, H., 2003 RNA interference: Traveling in the cell and gain- versely, post-transcriptional gene silencing by RNAi, in ing functions? Trends Genet. 19: 39–46. Cerutti, H., and J. A. Casas-Mollano, 2006 On the origin and both invertebrates and higher plants, appears to be functions of RNA-mediated silencing: from protists to man. Curr. more efficient at 25°–29° than at lower temperatures Genet. 50: 81–99. (Fortier and Belote 2000; Szittya et al. 2003). We Cerutti, H., A. M. Johnson,N.W.Gillham and J. E. Boynton, 1997a A eubacterial gene conferring spectinomycin resistance speculate that, in C. reinhardtii, multiple, partly indepen- on Chlamydomonas reinhardtii: integration into the nuclear ge- dent silencing mechanisms may allow a more effective nome and gene expression. Genetics 145: 97–110. control of transposon mobilization over a wide range of Cerutti, H., A. M. Johnson,N.W.Gillham and J. E. Boynton, environmental conditions. 1997b Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9: 925–945. Similarly to our observations in Chlamydomonas, the Cerutti, L., N. Mian and A. Bateman, 2000 Domains in gene silenc- LINE1 retrotransposon in humans and the P element in ing and cell differentiation proteins: the novel PAZ domain and redefinition of thePiwi domain.TrendsBiochem. Sci.25: 481–482. Drosophila appear to be repressed by multiple pathways han ilberman ie ohansen arrington oifer ossi ravin osse C , S. W., D. Z ,Z.X ,L.K.J ,J.C.C (S and R 2006; A et al. 2007; J et al. et al., 2004 RNA silencing genes control de novo DNA methyla- 2007). Since transposable elements are infectious tion. Science 303: 1336. hapman arrington agents in sexual populations, hosts are under selective C , E. J., and J. C. C , 2007 Specialization and evolution of endogenous small RNA pathways. Nat. Rev. Genet. 8: pressure to develop defensive functions that alleviate the 884–896. fitness penalty imposed by active transposons (Bestor Chicas, A., C. Cogoni and G. Macino, 2004 RNAi-dependent and 2003). Therefore, it is perhaps not surprising that RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa. Nucleic Acids Res. 32: eukaryotes seem to have evolved multiple epigenetic 4237–4243. silencing mechanisms, including specialized RNAi path- Cogoni, C., and G. Macino, 2000 Post-transcriptional gene silenc- ways, as a defense strategy against the massive expansion ing across kingdoms. Curr. Opin. Genet. Dev. 10: 638–643. Day, A., and J. D. Rochaix, 1991 Structure and inheritance of sense of transposable elements. Moreover, redundant, partly and anti-sense transcripts from a transposon in the green alga independent pathways likely increase the genetic ro- Chlamydomonas reinhardtii. J. Mol. Biol. 218: 273–291. bustness of transposon repression against mutational Day, A., M. Schirmer-Rahire,M.R.Kuchka,S.P.Mayfield and J. D. Rochaix, 1988 A transposon with an unusual arrangement of and environmental perturbations. long terminal repeats in the green alga Chlamydomonas reinhardtii. We thank Joint Genome Institute scientists for allowing access to the EMBO J. 7: 1917–1927. ing oinnet Volvox and Chlorella genome sequences prior to publication and D , S.-W., and O. V , 2007 Antiviral immunity directed by members of the Cerutti lab for critical reading of the manuscript. This small RNAs. Cell 130: 413–426. Dlakic, M., 2006 DUF283 domain of Dicer proteins has a double- work was supported by grants from the National Institutes of Health stranded RNA-binding fold. Bioinformatics 22: 2711–2714. and the National Science Foundation to H.C. We also acknowledge the Ferris, P.J., 1989 Characterization of a Chlamydomonas transposon, support of the Nebraska Experimental Program to Stimulate Com- Gulliver, resembling those in higher plants. Genetics 122: 363–377. petitive Research. Feschotte, C., N. Jiang and S. R. Wessler, 2002 Plant transposable elements: where genetics meets genomics. Nat. Rev. Genet. 3: LITERATURE CITED 329–341. Fire, A., S. Xu,M.K.Montgomery,S.A.Kostas,S.E.Driver et al., Allen, E., Z. Xie,A.M.Gustafson and J. C. Carrington, 1998 Potent and specific genetic interference by double- 2005 microRNA-directed phasing during trans-acting siRNA stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. biogenesis in plants. Cell 121: 207–221. Fortier, E., and J. M. Belote, 2000 Temperature-dependent gene Aoki, K., H. Moriguchi,T.Yoshioka,K.Okawa and H. Tabara, silencing by an expressed inverted repeat in Drosophila. Genesis 2007 In vitro analyses of the production and activity of second- 26: 240–244. ary small interfering RNAs in C. elegans. EMBO J. 26: 5007–5019. Freitag, M., D. W. Lee,G.O.Kothe,R.J.Pratt,R.Aramayo et al., Aravin, A. A., G. J. Hannon and J. Brennecke, 2007 The Piwi-piRNA 2004 DNA methylation is independent of RNA interference in pathway provides an adaptive defense in the transposon arms race. Neurospora. Science 304: 1939. Science 318: 761–764. Hall, J. L., and D. J. L. Luck, 1995 Basal body-associated DNA: in Bartel, D. P., 2004 MicroRNAs: genomics, biogenesis, mechanism, situ studies in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. and function. Cell 116: 282–297. USA 92: 5129–5133. 80 J. A. Casas-Mollano et al.

Harris, E. H., 1989 The Chlamydomonas Sourcebook. Academic Press, Nolan, T., L. Braccini,G.Azzalin,A.De Toni,G.Macino et al., San Diego. 2005 The post-transcriptional gene silencing machinery func- Hinas, A., J. Reimega˚rd,E.G.Wagner,W.Nellen,V.R.Ambros tions independently of DNA methylation to repress a LINE1-like et al., 2007 The small RNA repertoire of Dictyostelium discoideum retrotransposon in Neurospora crassa. Nucleic Acids Res. 33: 1564– and its regulation by components of the RNAi pathway. Nucleic 1573. Acids Res. 35: 6714–6726. Obbard, D. J., F. M. Jiggins,D.L.Halligan and T. J. Little, Ibrahim, F., J. Rohr, W-J. Jeong,J.Hesson and H. Cerutti, 2006 Natural selection drives extremely rapid evolution in an- 2006 Untemplated oligoadenylation promotes degradation of tiviral RNAi genes. Curr. Biol. 16: 580–585. RISC-cleaved transcripts. Science 314: 1893. Okamura, K., A. Ishizuka,H.Siomi and M. C. Siomi, 2004 Distinct Janowski, B. A., S. T. Younger,D.B.Hardy,R.Ram,K.E.Huffman roles for Argonaute proteins in small RNA-directed RNA cleavage et al., 2007 Activating gene expression in mammalian cells pathways. Genes Dev. 18: 1655–1666. with promoter-targeted duplex RNAs. Nat. Chem. Biol. 3: 166– Pak, J., and A. Fire, 2007 Distinct populations of primary and sec- 173. ondary effectors during RNAi in C. elegans. Science 315: 241–244. Jeong, B. R., D. Wu-Scharf,C.Zhang and H. Cerutti, 2002 Sup- Pe´rez-Alegre, M., A. Dubus and E. Fernańdez, 2005 REM1, a new pressors of transcriptional transgenic silencing in Chlamydomonas type of long terminal repeat transposon in Chlamydomonas rein- are sensitive to DNA-damaging agents and reactivate transpos- hardtii. Mol. Cell. Biol. 25: 10628–10638. able elements. Proc. Natl. Acad. Sci. USA 99: 1076–1081. Rangwala, S. H., and E. J. Richards, 2007 Differential epigenetic Josse, T.,L. Teysset,A.L.Todeschini,C.M.Sidor,D.Anxolabehere regulation within an Arabidopsis retroposon family. Genetics et al., 2007 Telomeric trans-silencing: an epigenetic repression 176: 151–160. combining RNA silencing and heterochromatin formation. PLoS Rivas, F. V., N. H. Tolia,J.J.Song,J.P.Aragon,J.Liu et al., Genet. 3: 1633–1643. 2005 Purified Argonaute2 and siRNA form recombinant hu- Keller, L. R., J. A. Schloss,C.D.Silflow and J. L. Rosenbaum, man RISC. Nat. Struct. Mol. Biol. 12: 340–349. 1984 Transcription of a- and b-tubulin genes in vitro in isolated Rohr, J., N. Sarkar,S.Balenger,B.R.Jeong and H. Cerutti, Chlamydomonas reinhardtii nuclei. J. Cell Biol. 98: 1138–1143. 2004 Tandem inverted repeat system for selection of effective Kim, K. S., S. Kustu and W. Inwood, 2006 Natural history of trans- transgenic RNAi strains in Chlamydomonas. Plant J. 40: 611–621. position in the green alga Chlamydomonas reinhardtii: use of the Rudenko, G. N., A. Ono and V. Walbot, 2003 Initiation of silencing AMT4 locus as an experimental system. Genetics 173: 2005–2019. of maize MuDR/Mu transposable elements. Plant J. 33: 1013– Kindle, K. L., 1990 High-frequency nuclear transformation of Chla- 1025. mydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87: 1228–1232. Saitou, N., and M. Nei, 1987 The neighbor-joining method: a new Kumar, S., K. Tamura and M. Nei, 2004 MEGA3: Integrated soft- method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: ware for Molecular Evolutionary Genetics Analysis and sequence 406–425. alignment. Brief. Bioinform. 2: 150–163. Sambrook, J., and D. W. Russell, 2001 Molecular Cloning: A Labora- Lee, S. R., and K. Collins, 2006 Two classes of endogenous small tory Manual. Cold Spring Harbor Laboratory Press, Cold Spring RNAs in Tetrahymena thermophila. Genes Dev. 20: 28–33. Harbor, NY. Li, L. C., S. T. Okino,H.Zhao,D.Pookot,R.F.Place et al., Schnell, R. A., and P. A. Lefebvre, 1993 Isolation of the Chlamy- 2006 Small dsRNAs induce transcriptional activation in human domonas regulatory gene NIT2 by transposon tagging. Genetics cells. Proc. Natl. Acad. Sci. USA 103: 17337–17342. 134: 737–747. Lippman, Z., and R. Martienssen, 2004 The role of RNA interfer- Schroda, M., 2006 RNA silencing in Chlamydomonas: mechanisms ence in heterochromatic silencing. Nature 431: 364–370. and tools. Curr. Genet. 49: 69–84. Lippman, Z., B. May,C.Yordan,T.Singer and R. Martienssen, Sijen, T., and R. H. Plasterk, 2003 Transposon silencing in the Cae- 2003 Distinct mechanisms determine transposon inheritance norhabditis elegans germ line by natural RNAi. Nature 426: 310– and methylation via small interfering RNA and histone modifica- 314. tion. PLoS Biol. 1: E67. Sijen, T., J. Fleenor,F.Simmer,K.L.Thijssen,S.Parrish et al., Liu, J., M. A. Carmell,F.V.Rivas,C.G.Marsden,J.M.Thomson 2001 On the role of RNA amplification in dsRNA-triggered et al., 2004 Argonaute2 is the catalytic engine of mammalian gene silencing. Cell 107: 465–476. RNAi. Science 305: 1437–1441. Slotkin, R. K., and R. Martienssen, 2007 Transposable elements Ma, J. B., K. Ye and D. J. Patel, 2004 Structural basis for overhang- and the epigenetic regulation of the genome. Nat. Rev. Genet. specific small interfering RNA recognition by the PAZ domain. 8: 272–285. Nature 429: 318–322. Soifer, H. S., and J. J. Rossi, 2006 Small interfering RNAs to the Margis, R., A. F. Fusaro,N.A.Smith,S.J.Curtin,J.M.Watson rescue: blocking L1 retrotransposition. Nat. Struct. Mol. Biol. et al., 2006 The evolution and diversification of Dicers in plants. 13: 758–759. FEBS Lett. 580: 2442–2450. Song, J. J., S. K. Smith,G.J.Hannon and L. Joshua-Tor, 2004 Crys- Matranga, C., and P. Zamore, 2007 Small silencing RNAs. Curr. tal structure of Argonaute and its implications for RISC slicer ac- Biol. 17: R789–R793. tivity. Science 305: 1434–1437. Matzke, M. A., and J. A. Birchler, 2005 RNAi-mediated pathways Stanek, D., and K. M. Neugebauer, 2006 The cajal body: a meeting in the nucleus. Nat. Rev. Genet. 6: 24–35. place for spliceosomal snRNPs in the nuclear maze. Chromosoma Matzke, M., T. Kanno,B.Huettel,L.Daxinger and A. J. Matzke, 115: 343–354. 2007 Targets of RNA-directed DNA methylation. Curr. Opin. Szittya, G., D. Silhavy,A.Molnar,Z.Havelda,A.Lovas et al., Plant Biol. 10: 512–519. 2003 Low temperature inhibits RNA silencing-mediated de- Meister, G., and T. Tuschl, 2004 Mechanisms of gene silencing by fence by the control of siRNA generation. EMBO J. 22: 633–640. double-stranded RNA. Nature 431: 343–349. Thompson, J. D., T. J. Gibson,F.Plewniak,F.Jeanmougin and D. G. Meister, G., M. Landthaler,A.Patkaniowska,Y.Dorsett,G. Higgins, 1997 The CLUSTAL_X windows interface: flexible Teng et al., 2004 Human Argonaute2 mediates RNA cleavage strategies for multiple sequence alignment aided by quality anal- targeted by miRNAs and siRNAs. Mol. Cell 15: 185–197. ysis tools. Nucleic Acids Res. 25: 4876–4882. Merchant, S. S., S. E. Prochnik,O.Vallon,E.H.Harris,S.J. Tran, R. K., D. Zilberman,C.De Bustos,R.F.Ditt,J.G.Henikoff Karpowicz et al., 2007 The Chlamydomonas genome reveals et al., 2005 Chromatin and siRNA pathways cooperate to main- the evolution of key animal and plant functions. Science 318: tain DNA methylation of small transposable elements in Arabidop- 245–251. sis. Genome Biol. 6: R90. Molnar, A., F. Schwach,D.J.Studholme,E.C.Thuenemann and van Dijk, K., K. E. Marley,B.R.Jeong,J.Xu,J.Hesson et al., D. C. Baulcombe, 2007 miRNAs control gene expression in 2005 Monomethyl histone H3 lysine 4 as an epigenetic mark the single-cell alga Chlamydomonas reinhardtii. Nature 447: 1126– for silenced euchromatin in Chlamydomonas. Plant Cell 17: 2439– 1129. 2453. Nakayashiki, H., N. Kadotani and S. Mayama, 2006 Evolution van Dijk, K., H. Xu and H. Cerutti, 2006 Epigenetic silencing of and diversification of RNA silencing proteins in fungi. J. Mol. transposons in the green alga Chlamydomonas reinhardtii. Nucleic Evol. 63: 127–135. Acids Mol. Biol. 17: 159–178. RNAi and Transposon Silencing 81

Vasudevan, S., Y. Tong and J. A. Steitz, 2007 Switching from re- Yuan, Y-R., Y. Pei, J-B. Ma,V.Kuryavyi,M.Zhadina et al., 2005 Crys- pression to activation: microRNAs can up-regulate translation. tal structure of A. aeolicus argonaute, a site-specific DNA-guided Science 318: 1931–1934. endoribonuclease, provides insights into RISC-mediated mRNA Vaucheret, H., 2006 Post-transcriptional small RNA pathways cleavage. Mol. Cell 19: 405–419. in plants: mechanisms and regulations. Genes Dev. 20: 759– Zambon, R. A., V. N. Vakharia and L. P. Wu, 2006 RNAi is an anti- 771. viral immune response against a dsRNA virus in Drosophila mela- Wang, S-C., R. A. Schnell and P. A. Lefebvre, 1998 Isolation and nogaster. Cell. Microbiol. 8: 880–889. characterization of a new transposable element in Chlamydomonas Zamore, P. D., and B. Haley, 2005 Ribo-gnome: the big world of reinhardtii. Plant Mol. Biol. 38: 681–687. small RNAs. Science 309: 1519–1524. Wienholds, E., and R. H. A. Plasterk, 2005 MicroRNA function in Zhang, C., D. Wu-Scharf,B.R.Jeong and H. Cerutti, 2002 A animal development. FEBS Lett. 579: 5911–5922. WD40-repeat containing protein, similar to a fungal co-repressor, Woodhouse, M. R., M. Freeling and D. Lisch, 2006 Initiation, es- is required for transcriptional gene silencing in Chlamydomonas. tablishment, and maintenance of heritable MuDR transposon si- Plant J. 31: 25–36. lencing in maize are mediated by distinct factors. PLoS Biol. 4: Zhao, T., G. Li,S.Mi,S.Li,G.J.Hannon et al., 2007 A complex e339. system of small RNAs in the unicellular green alga Chlamydomonas Xie, Z., L. K. Johansen,A.M.Gustafson,K.D.Kasschau,A.D. reinhardtii. Genes Dev. 21: 1190–1203. Lellis et al., 2004 Genetic and functional diversification of Zilberman, D., X. Cao and S. E. Jacobsen, 2003 Argonaute4 con- small RNA pathways in plants. PLoS Biol. 2: E104. trol of locus-specific siRNA accumulation and DNA and histone Yoshikawa, M., A. Peragine,M.Y.Park and R. S. Poethig, 2005 A methylation. Science 299: 716–719. pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19: 2164–2175. Communicating editor: S. Dutcher