Small RNAs >26 nt in length associate with AGO1 and are upregulated by nutrient deprivation in the alga Chlamydomonas.

Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in Chlamydomonas reinhardtii.

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

MLK4-mediated phosphorylation of histone H3T3 promotes flowering by transcriptional silencing of FLC/MAF in Arabidopsis thaliana.

Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9-LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with the transcriptional repression of euchromatic and heterochromatic loci. This study demonstrates that mlk4-3, a T-DNA insertion mutant of MLK4, flowered late, and that overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3T3 both in vitro and in vivo, and this catalytic activity required the conserved lysine residue K175. mutation of MLK4 at K175 failed to restore the level of phosphorylated H3T3 (H3T3ph) or to complement the phenotypic defects of mlk4-3. The FLC/MAF-clade genes, including FLC, MAF4 and MAF5, were significantly upregulated in mlk4-3. The double mutant mlk4-3 flc-3 flowered earlier than mlk4-3, suggesting that functional FLC is crucial for flowering repression in mlk4-3. Chromatin immunoprecipitation assays showed that MLK4 bound to FLC/MAF chromatin and that H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. In accordance, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4-3 but diminished in 35S::MLK4(K175R)/mlk4-3 plants. Moreover, the amount of RNA Pol II deposited at FLC/MAF was clearly enriched in mlk4-3 relative to the wild type. Therefore, MLK4-dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC/MAF. This study sheds light on the delicate control of flowering by the plant-specific CK1, MLK4, via post-translational modification of histone H3.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas.

Understanding the unique features of triacylglycerol (TAG) metabolism in microalgae may be necessary to realize the full potential of these organisms for biofuel and biomaterial production. In the unicellular green alga Chlamydomonas reinhardtii a chloroplastic (prokaryotic) pathway has been proposed to play a major role in TAG precursor biosynthesis. However, as reported here, C. reinhardtii contains a chlorophyte-specific lysophosphatidic acid acyltransferase, CrLPAAT2, that localizes to endoplasmic reticulum (ER) membranes. Unlike canonical, ER-located LPAATs, CrLPAAT2 prefers palmitoyl-CoA over oleoyl-CoA as the acyl donor substrate. RNA-mediated suppression of CrLPAAT2 indicated that the enzyme is required for TAG accumulation under nitrogen deprivation. Our findings suggest that Chlamydomonas has a distinct glycerolipid assembly pathway that relies on CrLPAAT2 to generate prokaryotic-like TAG precursors in the ER.

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii.

Canonical microRNAs (miRNAs) are embedded in duplexed stem-loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

A multidomain enzyme, with glycerol-3-phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii.

Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green alga C. reinhardtii showed substantial triacylglycerol (TAG) accumulation and up-regulation of a gene, GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol-3-phosphate dehydrogenase (GPD) domains. Canonical GPD enzymes catalyze the synthesis of glycerol-3-phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone of TAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. Recombinant Chlamydomonas GPD2 showed both reductase and phosphatase activities in vitro and it can work as a bifunctional enzyme capable of synthesizing glycerol directly from DHAP. In addition, GPD2 and a gene encoding glycerol kinase were up-regulated in Chlamydomonas cells exposed to high salinity. RNA-mediated silencing of GPD2 revealed that the multidomain enzyme was required for TAG accumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, a GPD2-mCherry fusion protein was found to localize to the chloroplast, supporting the existence of a GPD2-dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities of PSP-GPD multidomain enzymes may be modulated by post-translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana.

Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5' genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).

Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas.

MicroRNAs (miRNAs) play important roles in diverse biological processes in eukaryotes, generally through degradation and/or inhibition of the translation of target mRNAs. MicroRNAs are loaded into Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC) and used as guides to identify complementary transcripts. The distinct functions and features, such as associated small RNA classes and modes of silencing, of individual AGO paralogs have been well documented in multicellular eukaryotes. However, this aspect of miRNA function remains poorly understood in the unicellular green alga Chlamydomonas reinhardtii, which contains three AGO paralogs. In this study, we isolated AGO2 and AGO3 insertional mutants and confirmed that AGO3 is more abundantly expressed than AGO2. MicroRNA-directed target transcript cleavage and translational repression were impaired in the AGO3 mutant background, indicating that AGO3 can mediate both modes of silencing. In contrast, although the AGO2 mutant is not a null, the involvement of AGO2 in miRNA-directed silencing appears to be more limited. Our results strongly suggest that miRNA-mediated post-transcriptional gene silencing relies primarily on AGO3 in Chlamydomonas.

SUMOylation by a stress-specific small ubiquitin-like modifier E2 conjugase is essential for survival of Chlamydomonas reinhardtii under stress conditions.

Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) is required for survival of virtually all eukaryotic organisms. Attachment of SUMO to target proteins is catalyzed by SUMO E2 conjugase. All haploid or diploid eukaryotes studied to date possess a single indispensable SUMO conjugase. We report here the unanticipated isolation of a Chlamydomonas reinhardtii (mutant5 [mut5]). in which the previously identified SUMO conjugase gene C. reinhardtii ubiquitin-conjugating enzyme9 (CrUBC9) is deleted. This surprising mutant is viable and unexpectedly, displays a pattern of protein SUMOylation at 25°C that is essentially identical to wild-type cells. However, unlike wild-type cells, mut5 fails to SUMOylate a large set of proteins in response to multiple stress conditions, a failure that results in a markedly reduced tolerance or complete lack of tolerance to these stresses. Restoration of expected stress-induced protein SUMOylation patterns as well as normal stress tolerance phenotypes in mut5 cells complemented with a CrUBC9 gene shows that CrUBC9 is an authentic SUMO conjugase and, more importantly, that SUMOylation is essential for cell survival under stress conditions. The presence of bona fide SUMOylated proteins in the mut5 mutant at 25°C can only be explained by the presence of at least one additional SUMO conjugase in C. reinhardtii, a conjugase tentatively identified as CrUBC3. Together, these results suggest that, unlike all other nonpolyploid eukaryotes, there are at least two distinct and functional SUMO E2 conjugases in C. reinhardtii, with a clear division of labor between the two sets: One (CrUBC9) is involved in essential stress-induced SUMOylations, and one (CrUBC3) is involved in housekeeping SUMOylations.

Small interfering RNA-mediated translation repression alters ribosome sensitivity to inhibition by cycloheximide in Chlamydomonas reinhardtii.

Small RNAs (sRNAs; ∼20 to 30 nucleotides in length) play important roles in gene regulation as well as in defense responses against transposons and viruses in eukaryotes. Their biogenesis and modes of action have attracted great attention in recent years. However, many aspects of sRNA function, such as the mechanism(s) of translation repression at postinitiation steps, remain poorly characterized. In the unicellular green alga Chlamydomonas reinhardtii, sRNAs derived from genome-integrated inverted repeat transgenes, perfectly complementary to the 3' untranslated region of a target transcript, can inhibit protein synthesis without or with only minimal mRNA destabilization. Here, we report that the sRNA-repressed transcripts are not altered in their polyadenylation status and they remain associated with polyribosomes, indicating inhibition at a postinitiation step of translation. Interestingly, ribosomes associated with sRNA-repressed transcripts show reduced sensitivity to translation inhibition by some antibiotics, such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in C. reinhardtii may involve alterations to the function/structural conformation of translating ribosomes. Additionally, sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, suggesting that this mechanism may have been a feature of an ancestral RNA interference machinery.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

Recurrent evolutionary switches of mitochondrial cytochrome c maturation systems in Archaeplastida.

Mitochondrial cytochrome c maturation (CCM) requires heme attachment via distinct pathways termed systems I and III. The mosaic distribution of these systems in Archaeplastida raises questions about the genetic mechanisms and evolutionary forces promoting repeated evolution. Here, we show a recurrent shift from ancestral system I to the eukaryotic-specific holocytochrome c synthase (HCCS) of system III in 11 archaeplastid lineages. Archaeplastid HCCS is sufficient to rescue mutants of yeast system III and Arabidopsis system I. Algal HCCS mutants exhibit impaired growth and respiration, and altered biochemical and metabolic profiles, likely resulting from deficient CCM and reduced cytochrome c-dependent respiratory activity. Our findings demonstrate that archaeplastid HCCS homologs function as system III components in the absence of system I. These results elucidate the evolutionary trajectory and functional divergence of CCM pathways in Archaeplastida, providing insight into the causes, mechanisms, and consequences of repeated cooption of an entire biological pathway.

Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas.

Regulation of gene expression by small RNAs ( approximately 20-30 nucleotides in length) plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) commonly direct the inactivation of cognate sequences through a variety of mechanisms, including RNA degradation, translation inhibition, and transcriptional repression. Recent studies have provided considerable insight into the biogenesis and the mode of action of miRNAs and siRNAs. However, relatively little is known about mechanisms of quality control and small RNA decay in RNA interference (RNAi) pathways. Here we show that deletion of MUT68, encoding a terminal nucleotidyltransferase in the alga Chlamydomonas reinhardtii, results in elevated miRNA and siRNA levels. We found that MUT68 plays a role in the untemplated uridylation of the 3' ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Moreover, RRP6 depletion also leads to accumulation of small RNAs in vivo. We propose that MUT68 and RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as a quality control mechanism to eliminate dysfunctional or damaged small RNA molecules.

RNA-mediated silencing in Algae: biological roles and tools for analysis of gene function.

Algae are a large group of aquatic, typically photosynthetic, eukaryotes that include species from very diverse phylogenetic lineages, from those similar to land plants to those related to protist parasites. The recent sequencing of several algal genomes has provided insights into the great complexity of these organisms. Genomic information has also emphasized our lack of knowledge of the functions of many predicted genes, as well as the gene regulatory mechanisms in algae. Core components of the machinery for RNA-mediated silencing show widespread distribution among algal lineages, but they also seem to have been lost entirely from several species with relatively small nuclear genomes. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have now been identified by high-throughput sequencing in green, red, and brown algae. However, the natural roles of RNA-mediated silencing in algal biology remain poorly understood. Limited evidence suggests that small RNAs may function, in different algae, in defense mechanisms against transposon mobilization, in responses to nutrient deprivation and, possibly, in the regulation of recently evolved developmental processes. From a practical perspective, RNA interference (RNAi) is becoming a promising tool for assessing gene function by sequence-specific knockdown. Transient gene silencing, triggered with exogenously synthesized nucleic acids, and/or stable gene repression, involving genome-integrated transgenes, have been achieved in green algae, diatoms, yellow-green algae, and euglenoids. The development of RNAi technology in conjunction with system level "omics" approaches may provide the tools needed to advance our understanding of algal physiological and metabolic processes.

Turnover of Mature miRNAs and siRNAs in Plants and Algae.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) play important roles in gene regulation and defense responses against transposons and viruses in eukaryotes. These small RNAs generally trigger the silencing of cognate sequences through a variety of mechanisms, including RNA degradation, translational inhibition and transcriptional repression. In the past few years, the synthesis and the mode of action of miRNAs and siRNAs have attracted great attention. However, relatively little is known about mechanisms of quality control during small RNA biogenesis as well as those that regulate mature small RNA stability. Recent studies in Arabidopsis thaliana and Caenorhabditis elegans have implicated 3'-to-5' (SDNs) and 5'-to-3' (XRN-2) exoribonucleases in mature miRNA turnover and the modulation of small RNA levels and activity. In the green alga Chlamydomonas reinhardtii, a nucleotidyltransferase (MUT68) and an exosome subunit (RRP6) are involved in the 3' untemplated uridylation and the degradation of miRNAs and siRNAs. The latter enzymes appear to function as a quality control mechanism to eliminate putative dysfunctional or damaged small RNA molecules. Several post-transcriptional modifications of miRNAs and siRNAs such as 3' terminal methylation and untemplated nucleotide additions have also been reported to affect small RNA stability. These collective findings are beginning to uncover a new layer of regulatory control in the pathways involving small RNAs. We anticipate that understanding the mechanisms of mature miRNA and siRNA turnover will have direct implications for fundamental biology as well as for applications of RNA interference technology.

The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas.

Changes in chromatin organization are emerging as key regulators in nearly every aspect of DNA-templated metabolism in eukaryotes. Histones undergo many, largely reversible, posttranslational modifications that affect chromatin structure. Some modifications, such as trimethylation of histone H3 on Lys 4 (H3K4me3), correlate with transcriptional activation, whereas others, such as methylation of histone H3 on Lys 27 (H3K27me), are associated with silent chromatin. Posttranslational histone modifications may also be involved in the inheritance of chromatin states. Histone phosphorylation has been implicated in a variety of cellular processes but, because of the dynamic nature of this modification, its potential role in long-term gene silencing has remained relatively unexplored. We report here that a Chlamydomonas reinhardtii mutant defective in a Ser/Thr protein kinase (MUT9p), which phosphorylates histones H3 and H2A, shows deficiencies in the heritable repression of transgenes and transposons. Moreover, based on chromatin immunoprecipitation analyses, phosphorylated H3T3 (H3T3ph) and monomethylated H3K4 (H3K4me1) are inversely correlated with di/trimethylated H3K4 and associate preferentially with silenced transcription units. Conversely, the loss of those marks in mutant strains correlates with the transcriptional reactivation of transgenes and transposons. Our results suggest that H3T3ph and H3K4me1 function as reinforcing epigenetic marks for the silencing of euchromatic loci in Chlamydomonas.

Turnover of mature miRNAs and siRNAs in plants and algae.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) play important roles in gene regulation and defense responses against transposons and viruses in eukaryotes. These small RNAs generally trigger the silencing of cognate sequences through a variety of mechanisms, including RNA degradation, translational inhibition and transcriptional repression. In the past few years, the synthesis and the mode of action of miRNAs and siRNAs have attracted great attention. However, relatively little is known about mechanisms of quality control during small RNA biogenesis as well as those that regulate mature small RNA stability. Recent studies in Arabidopsis thaliana and Caenorhabditis elegans have implicated 3'-to-5' (SDNs) and 5'-to-3' (XRN-2) exoribonucleases in mature miRNA turnover and the modulation of small RNA levels and activity. In the green alga Chlamydomonas reinhardtii, a nucleotidyltransferase (MUT68) and an exosome subunit (RRP6) are involved in the 3' untemplated uridylation and the degradation of miRNAs and siRNAs. The latter enzymes appear to function as a quality control mechanism to eliminate putative dysfunctional or damaged small RNA molecules. Several post-transcriptional modifications of miRNAs and siRNAs such as 3' terminal methylation and untemplated nucleotide additions have also been reported to affect small RNA stability. These collective findings are beginning to uncover a new layer of regulatory control in the pathways involving small RNAs. We anticipate that understanding the mechanisms of mature miRNA and siRNA turnover will have direct implications for fundamental biology as well as for applications of RNA interference technology.