Diversification of the core RNA interference machinery in Chlamydomonas reinhardtii and the role of DCL1 in transposon silencing

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.

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).

Figure 2.—

Phylogenetic tree of Dicer-like proteins. Sequences corresponding to the RNaseIII domains were aligned using the ClustalX program, and a neighbor-joining tree was constructed using MEGA v3.1. Numbers show 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, as described in the legend to Figure 1. Accession numbers of proteins used to draw the tree are the following: At DCL1, NP_171612; At DCL2, NP_566199; At DCL3, NP_189978; At DCL4, NP_197532; Cr DCL1, EU368690; Cr DCL2, XP_001698921; Cr DCL3, XP_001692436; Cs NC64A scaffold-5-57 gene7, v2_NC64A_scaffold_5_57_gene7; Dm Dcr1, Q9VCU9; Dm Dcr2, BAB69959; Hs DCR1, NP_803187; Os DCL1, NP_912466; Os DCL2a, XP_463068; Os DCL2b, BAD34005; Os DCL3a, XP_463595; Os DCL3b, NP_922059; Os DCL4, XP_473129; Vc_106340, v1_106340. Accession numbers in italic 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).

Figure 3.—

Genomic organization of C. reinhardtii DCL1 and AGO1 and schematic 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 5′ and 3′ 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.

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).

Figure 5.—

DCL1 is dispensable for transcriptional silencing of the TOC1 retrotransposon. (A) Transcriptional activity of TOC1 in nuclear run-on assays of the indicated strains, described in the legend to Figure 4. Transcription of TUBA (encoding α-tubulin) and ACT1 (encoding actin) was evaluated as a control. (B) Southern blot analysis of TOC1 and GULLIVER cytosine DNA methylation with isoschizomeric restriction 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/MspI or XbaI/HpaII cleavage would result in a GULLIVER fragment of 210 bp. The multiple segments obtained with HinfI or XbaI digestions reflect sequence heterogeneity among different copies of TOC1 or GULLIVER. (C) Immunoblot 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 ±SD of three independent experiments.

Figure 6.—

Subcellular compartmentalization of TOC1 transcripts and siRNAs. (A) Northern blot analysis of TOC1 long RNAs in whole cells (WHOLE), the cytoplasmic fraction (CYT), or the nuclear fraction (NUC) from a cell-wall-less strain (CC-3491). Purified RNAs were resolved on agarose–formaldehyde gels and hybridized to a TOC1 probe. The same blot was reprobed for ACT1, a predominantly cytoplasmic transcript, and for the U6 small nuclear RNA, retained in the nucleus, to assess the effectiveness of the cell fractionation. (B) Northern blot detection of TOC1 siRNAs in the different subcellular fractions. Column-purified small RNAs were separated in a 15% denaturing polyacrylamide gel, electroblotted onto a nylon membrane, and sequentially hybridized with probes corresponding to the TOC1 LTR and the U6 small nuclear RNA.