Valérie Cognat

Coordination of Nuclear and Mitochondrial Genome Expression during Mitochondrial Biogenesis in Arabidopsis

Mitochondrial biogenesis and function require the regulated and coordinated expression of nuclear and mitochondrial genomes throughout plant development and in response to cellular and environmental signals. To investigate the levels at which the expression of nuclear and mitochondrially encoded proteins is coordinated, we established an Arabidopsis thaliana cell culture system to modulate mitochondrial biogenesis in response to sugar starvation and refeeding. Sucrose deprivation led to structural changes in mitochondria, a decrease in mitochondrial volume, and a reduction in the rate of cellular respiration. All these changes could be reversed by the readdition of sucrose. Analysis of the relative mRNA transcript abundance of genes encoding nuclear and mitochondrially encoded proteins revealed that there was no coordination of expression of the two genomes at the transcript level. An analysis of changes in abundance and assembly of nuclear-encoded and mitochondrially encoded subunits of complexes I to V of the mitochondrial inner membrane in organello protein synthesis and competence for protein import by isolated mitochondria suggested that coordination occurs at the level of protein-complex assembly. These results further suggest that expression of the mitochondrial genome is insensitive to the stress imposed by sugar starvation and that mitochondrial biogenesis is regulated by changes in nuclear gene expression and coordinated at the posttranslational level.

The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana

Post-translational modifications of proteins by addition of ubiquitin can regulate protein degradation and localization, protein-protein interactions and transcriptional activation. In the ubiquitylation system, substrate specificity is primarily determined by the E2 ubiquitin-conjugating enzyme (UBC) and the E3 ubiquitin ligase. The Arabidopsis thaliana genome contains 37 genes encoding UBC homologs. However, the biological functions of these genes remain largely uncharacterized. Here, we report reverse genetic characterization of AtUBC1 and AtUBC2. While the loss-of-function single mutants Atubc1-1 and Atubc2-1 only show weak phenotypes, the double mutant Atubc1-1 Atubc2-1 shows a dramatically reduced number of rosette leaves and an early-flowering phenotype. Consistent with these results, the transcript levels of the floral repressor genes FLOWERING LOCUS C (FLC), MADS ASSOCIATED FLOWERING 4 (MAF4) and MAF5 are reduced in the double mutant. Loss-of-function mutants of HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, which were previously reported to encode an E3 involved in histone H2B ubiquitylation, also show an early-flowering phenotype and reduced levels of FLC, MAF4 and MAF5 transcripts. In both Atubc1-1 Atubc2-1 and hub2-2 mutants, H2B mono-ubiquitylation is drastically reduced. Taken together, our results indicate that E2s AtUBC1/AtUBC2 and E3s HUB1/HUB2 together mediate H2B ubiquitylation, which is involved in the activation of floral repressor genes as well as in other processes as indicated by the pleiotropic phenotypes of the mutants.

Mouse Cytomegalovirus MicroRNAs Dominate the Cellular Small RNA Profile during Lytic Infection and Show Features of Posttranscriptional Regulation

ABSTRACT MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals, miRNAs have recently been identified in several viruses. Viral miRNAs may play a role in modulating both viral and host gene expression. Here, we report on the identification and characterization of 18 viral miRNAs from mouse fibroblasts lytically infected with the murine cytomegalovirus (MCMV). The MCMV miRNAs are expressed at early times of infection and are scattered in small clusters throughout the genome with up to four distinct miRNAs processed from a single transcript. No significant homologies to human CMV-encoded miRNAs were found. Remarkably, as soon as 24 h after infection, MCMV miRNAs constituted about 35% of the total miRNA pool, and at 72 h postinfection, this proportion was increased to more than 60%. However, despite the abundance of viral miRNAs during the early phase of infection, the expression of some MCMV miRNAs appeared to be regulated. Hence, for three miRNAs we observed polyuridylation of their 3′ end, coupled to subsequent degradation. Individual knockout mutants of two of the most abundant MCMV miRNAs, miR-m01-4 and miR-M44-1, or a double knockout mutant of miR-m21-1 and miR-M23-2, incurred no or only a very mild growth deficit in murine embryonic fibroblasts in vitro.

Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

SET DOMAIN GROUP25 Encodes a Histone Methyltransferase and Is Involved in FLOWERING LOCUS C Activation and Repression of Flowering

Covalent modifications of histone lysine residues by methylation play key roles in the regulation of chromatin structure and function. In contrast to H3K9 and H3K27 methylations that mark repressive states of transcription and are absent in some lower eukaryotes, H3K4 and H3K36 methylations are considered as active marks of transcription and are highly conserved in all eukaryotes from yeast (Saccharomyces cerevisiae) to Homo sapiens. Paradoxically, protein complexes catalyzing H3K4 and H3K36 methylations are less-extensively characterized in higher eukaryotes, particularly in plants. Arabidopsis (Arabidopsis thaliana) contains 12 SET DOMAIN GROUP (SDG) proteins phylogenetic classified to Trithorax Group (TrxG) and thus potentially involved in H3K4 and H3K36 methylations. So far only some genes of this family had been functionally characterized. Here we report on the genetic and molecular characterization of SDG25, a previously uncharacterized member of the Arabidopsis TrxG family. We show that the loss-of-function mutant sdg25-1 has an early flowering phenotype associated with suppression of FLOWERING LOCUS C (FLC) expression. Recombinant SDG25 proteins could methylate histone H3 from oligonucleosomes and mutant sdg25-1 plants showed weakly reduced levels of H3K36 dimethylation at FLC chromatin. Interestingly, sdg25-1 transcriptome shared a highly significant number of differentially expressed genes with that of sdg26-1, a previously characterized mutant exhibiting late-flowering phenotype and elevated FLC expression. Taken together, our results provide, to our knowledge, the first demonstration for a biological function of SDG25 and reveal additional layers of complexity of overlap and nonoverlap functions of the TrxG family genes in Arabidopsis.

Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana.

ABSTRACT Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Δ strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5′ part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

Two MicroRNAs Linked to Nodule Infection and Nitrogen-Fixing Ability in the Legume Lotus japonicus

Legumes overcome nitrogen shortage by developing root nodules in which symbiotic bacteria fix atmospheric nitrogen in exchange for host-derived carbohydrates and mineral nutrients. Nodule development involves the distinct processes of nodule organogenesis, bacterial infection, and the onset of nitrogen fixation. These entail profound, dynamic gene expression changes, notably contributed to by microRNAs (miRNAs). Here, we used deep-sequencing, candidate-based expression studies and a selection of Lotus japonicus mutants uncoupling different symbiosis stages to identify miRNAs involved in symbiotic nitrogen fixation. Induction of a noncanonical miR171 isoform, which targets the key nodulation transcription factor Nodulation Signaling Pathway2, correlates with bacterial infection in nodules. A second candidate, miR397, is systemically induced in the presence of active, nitrogen-fixing nodules but not in that of noninfected or inactive nodule organs. It is involved in nitrogen fixation-related copper homeostasis and targets a member of the laccase copper protein family. These findings thus identify two miRNAs specifically responding to symbiotic infection and nodule function in legumes.

The Arabidopsis CUL4–DDB1 complex interacts with MSI1 and is required to maintain MEDEA parental imprinting

Protein ubiquitylation regulates a broad variety of biological processes in all eukaryotes. Recent work identified a novel class of cullin‐containing ubiquitin ligases (E3s) composed of CUL4, DDB1, and one WD40 protein, believed to act as a substrate receptor. Strikingly, CUL4‐based E3 ligases (CRL4s) have important functions at the chromatin level, including responses to DNA damage in metazoans and plants and, in fission yeast, in heterochromatin silencing. Among putative CRL4 receptors we identified MULTICOPY SUPPRESSOR OF IRA1 (MSI1), which belongs to an evolutionary conserved protein family. MSI1‐like proteins contribute to different protein complexes, including the epigenetic regulatory Polycomb repressive complex 2 (PRC2). Here, we provide evidence that Arabidopsis MSI1 physically interacts with DDB1A and is part of a multimeric protein complex including CUL4. CUL4 and DDB1 loss‐of‐function lead to embryo lethality. Interestingly, as in fis class mutants, cul4 mutants exhibit autonomous endosperm initiation and loss of parental imprinting of MEDEA, a target gene of the Arabidopsis PRC2 complex. In addition, after pollination both MEDEA transcript and protein accumulate in a cul4 mutant background. Overall, our work provides the first evidence of a physical and functional link between a CRL4 E3 ligase and a PRC2 complex, thus indicating a novel role of ubiquitylation in the repression of gene expression.

A global picture of tRNA genes in plant genomes

Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for tRNA(Tyr) and tRNA(Mete) . By contrast, in green algae, more than half of the tRNA genes contain an intron. This suggests divergent means of intron acquisition and the splicing process between green algae and land plants. Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in flowering plants. The only exceptions are tRNA(Gly) -snoRNA and tRNA(Mete) -snoRNA cotranscripts in dicots and monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and plastidial tRNA gene populations is also provided.

Ribonucleotide Reductase Regulation in Response to Genotoxic Stress in Arabidopsis

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.