Genetic regulation of life cycle transitions in the brown alga Ectocarpus

The life cycle of an organism is one of its most elemental features, underpinning a broad range of phenomena including developmental processes, reproductive fitness, mode of dispersal and adaptation to the local environment. Life cycle modification may have played an important role during the evolution of several eukaryotic groups, including the terrestrial plants. Brown algae are potentially interesting models to study life cycle evolution because this group exhibits a broad range of different life cycles. Currently, life cycle studies are focused on the emerging brown algal model Ectocarpus. Two life cycle mutants have been described in this species, both of which cause the sporophyte generation to exhibit gametophyte characteristics. The ouroboros mutation is particularly interesting because it induces complete conversion of the sporophyte generation into a functional, gamete-producing gametophyte, a class of mutation that has not been described so far in other systems. Analysis of Ectocarpus life cycle mutants is providing insights into several life-cycle-related processes including parthenogenesis, symmetric/asymmetric initial cell divisions and sex determination.     

microRNAs in a multicellular green alga Volvox carteri

microRNAs(miRNAs)have emerged as key components in the eukaryotic gene regulatory network.We and others have previously identified many miRNAs in a unicellular green alga,Chlamydomonas reinhardtii.To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage,we examined small RNAs in Volvox carteri,a multicellular species in the same family with Chlamydomonas reinhardtii.We identified 174 miRNAs in Volvox,with many of them being highly enriched in gonidia or somatic cells.The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo,suggesting that miRNAs play regulatory roles in the biology of green algae.Our catalog of miRNAs and their targets provides a resource for further studies on the evolution,biological functions,and genomic properties of miRNAs in green algae.     

Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus

We used an in silico approach to predict microRNAs (miRNAs) genome-wide in the brown alga Ectocarpus siliculosus. As brown algae are phylogenetically distant from both animals and land plants, our approach relied on features shared by all known organisms, excluding sequence conservation, genome localization and pattern of base-pairing with the target. We predicted between 500 and 1500 miRNAs candidates, depending on the values of the energetic parameters used to filter the potential precursors. Using quantitative polymerase chain reaction assays, we confirmed the existence of 22 miRNAs among 72 candidates tested, and of 8 predicted precursors. In addition, we compared the expression of miRNAs and their precursors in two life cycle states (sporophyte, gametophyte) and under salt stress. Several miRNA precursors, Argonaute and DICER messenger RNAs were differentially expressed in these conditions. Finally, we analyzed the gene organization and the target functions of the predicted candidates. This showed that E. siliculosus miRNA genes are, like plant miRNA genes, rarely clustered and, like animal miRNA genes, often located in introns. Among the predicted targets, several widely conserved functional domains are significantly overrepresented, like kinesin, nucleotide-binding/APAF-1, R proteins and CED-4 (NB-ARC) and tetratricopeptide repeats. The combination of computational and experimental approaches thus emphasizes the originality of molecular and cellular processes in brown algae.     

The diversity and phylogeny of the commercially important algal class Eustigmatophyceae,including the new clade Goniochloridales

The Eustigmatophyceae is a class of yellow-green algae allied with the Chrysophyceae and other chlorophyll c possessing stramenopile (heterokont) algae. Some members of the class, especially the marine species of the genus Nannochloropsis, are under intense investigation for their potential for production of biofuels and beneficial fatty acids. The class has generally been thought to comprise a small number of genera and species, and these organisms were considered rare or infrequently encountered. In this study, we examined the phylogeny and diversity of this class by analysis of nuclear 18S rDNA sequence data. Our analysis included sequences from all the named members of the Eustigmatophyceae held in culture collections as well as a number of strains identified in culture collections as Xanthophyceae, new strains with features characteristic of the Eustigmatophyceae, and published data for uncultured DNA clones. The results of these analyses show that the Eustigmatophyceae is far more diverse than generally recognized. Two major lineages are supported in the class, the previously recognized order Eustigmatales and the new clade, Goniochloridales. Additional new lineages were also resolved within each of these major lineages; however, the results of our analyses were considered insufficient for naming these subordinate clades. Several of these lineages comprised only unnamed strains or uncultured DNA clones. Overall, our results indicate that the Eustigmatophyceae is a highly diverse class, with many new species, genera, and families awaiting taxonomic treatment.     

Identification of microRNAs and their targets from Populus euphratica

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.     

Development of PCR‐Based Markers to Determine the Sex of Kelps

Sex discriminating genetic markers are commonly used to facilitate breeding programs in economically and ecologically important animal and plant species. However, despite their considerable economic and ecological value, the development of sex markers for kelp species has been very limited. In this study, we used the recently described sequence of the sex determining region (SDR) of the brown algal model Ectocarpus to develop novel DNA-based sex-markers for three commercially relevant kelps: Laminaria digitata, Undaria pinnatifida and Macrocystis pyrifera. Markers were designed within nine protein coding genes of Ectocarpus male and female (U/V) sex chromosomes and tested on gametophytes of the three kelp species. Seven primer pairs corresponding to three loci in the Ectocarpus SDR amplified sex-specific bands in the three kelp species, yielding at least one male and one female marker for each species. Our work has generated the first male sex-specific markers for L. digitata and U. pinnatifida, as well as the first sex markers developed for the genus Macrocystis. The markers and methodology presented here will not only facilitate seaweed breeding programs but also represent useful tools for population and demography studies and provide a means to investigate the evolution of sex determination across this largely understudied eukaryotic group.     

Sex-Biased Expression of MicroRNAs in Schistosoma mansoni

Schistosomiasis is an important neglected tropical disease caused by digenean helminth parasites of the genus Schistosoma. Schistosomes are unusual in that they are dioecious and the adult worms live in the blood system. MicroRNAs play crucial roles during gene regulation and are likely to be important in sex differentiation in dioecious species. Here we characterize 112 microRNAs from adult Schistosoma mansoni individuals, including 84 novel microRNA families, and investigate the expression pattern in different sexes. By deep sequencing, we measured the relative expression levels of conserved and newly identified microRNAs between male and female samples. We observed that 13 microRNAs exhibited sex-biased expression, 10 of which are more abundant in females than in males. Sex chromosomes showed a paucity of female-biased genes, as predicted by theoretical evolutionary models. We propose that the recent emergence of separate sexes in Schistosoma had an effect on the chromosomal distribution and evolution of microRNAs, and that microRNAs are likely to participate in the sex differentiation/maintenance process.     

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis—a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.     

Rac1 function during fucoid development

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.Key words: actin, Arp2/3 complex, manumycin A, NSC23766, Rac1, Rho GTPase, Scar/WAVE, Silvetia compressa     

Target Repression Induced by Endogenous microRNAs: Large Differences,Small Effects

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.     

microRNAs in Circulation Are Altered in Response to Influenza A Virus Infection in Humans

Changes in microRNA expression have been detected in vitro in influenza infected cells, yet little is known about them in patients. microRNA profiling was performed on whole blood of H1N1 patients to identify signature microRNAs to better understand the gene regulation involved and possibly improve diagnosis. Total RNA extracted from blood samples of influenza infected patients and healthy controls were subjected to microRNA microarray. Expression profiles of circulating microRNAs were altered and distinctly different in influenza patients. Expression of highly dysregulated microRNAs were validated using quantitative PCR. Fourteen highly dysregulated miRNAs, identified from the blood of influenza infected patients, provided a clear distinction between infected and healthy individuals. Of these, expression of miR-1260, -26a, -335*, -576-3p, -628-3p and -664 were consistently dysregulated in both whole blood and H1N1 infected cells. Potential host and viral gene targets were identified and the impact of microRNA dysregulation on the host proteome was studied. Consequences of their altered expression were extrapolated to changes in the host proteome expression. These highly dysregulated microRNAs may have crucial roles in influenza pathogenesis and are potential biomarkers of influenza.