Characterization of the small RNAs of Volvox carteri
ABSTRACT: The green alga Volvox carteri is a model organism for the development of multicellularity. It has a spherical shape with a complete division of labor between around 2000 somatic cells and 16 reproductive cells. When comparing Volvox with its unicellular relative Chlamydomonas rheinhardtii, one striking observation is the similarity in the protein coding genes . Additionally, Baulcombe and colleagues showed that Chlamydomonas contains functional RNAi and miRNA machineries . We deep sequenced small RNAs of the female Volvox strain HK10 in different life stages (asexual reproduction to sexual reproduction), each time dividing the samples into somatic cells and reproductive cells. This allowed for the observation not only of differences in individual life stages, but also for monitoring sRNA content in the two cell types. We show that Volvox expresses miRNAs and that they are 2’-O-methylated at the 3’ end. The expression profiles of several miRNAs were validated by Northern blotting showing a differential expression both between cell types and between life stages. Intriguingly, most miRNAs do not seem to be conserved between Volvox and Chlamydomonas, raising the interesting question if this changed miRNome leads to differently targeted mRNAs thus resulting in cell differentiation. Since only little is known about the transcriptome of Volvox, we performed RNASeq in order to analyze potential miRNA targets. In conclusion, most miRNA in Volvox are not conserved in Chlamydomonas although the two species are evolutionary close together. This suggests that dramatic changes in the miRNA expression might be one of the driving forces for the development of multicellularity. 1. Prochnik, S.E., et al., Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science, 2010. 329(5988): p. 223-6. 2. Molnar, A., et al., miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature, 2007. 447(7148): p. 1126-9. Examination of small RNAs of Volvox carteri during different stages of its life cycle
Project description:The green alga Volvox carteri is a model organism for the development of multicellularity. It has a spherical shape with a complete division of labor between around 2000 somatic cells and 16 reproductive cells. When comparing Volvox with its unicellular relative Chlamydomonas rheinhardtii, one striking observation is the similarity in the protein coding genes . Additionally, Baulcombe and colleagues showed that Chlamydomonas contains functional RNAi and miRNA machineries . We deep sequenced small RNAs associated with one Argonaute protein of the female Volvox strain Vol6 during its vegetative growth phase. Using these data, we established a miRNA identification pipeline that takes into account plant miRNA feature in general and also uses parameters employed in finding miRNAs in Chlamydomonas. Other small RNAs that are functionally incorporated into Ago are characterized. 1. Prochnik, S.E., et al., Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science, 2010. 329(5988): p. 223-6. 2. Molnar, A., et al., miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature, 2007. 447(7148): p. 1126-9. Examination of small RNAs bound to an Argonaute protein of Volvox carteri
Project description:Motivation: MicroRNAs (miRNAs) are short regulatory RNAs derived from a longer precursor RNA. miRNA biogenesis has been studied in animals and plants, recently elucidating more diverse and complex aspects, such as non-conserved, speciesspecific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can be used to computationally determine genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage: while the mature miRNA typically produces hundreds of sequence reads, the remaining part of the precursor is covered very sparsely. Results: We introduce a new conservation-independent method for the identification of miRNA precursors, that allows for speciesspecific heterogeneous precursor populations. The algorithm requires small RNA sequencing data and evaluates precursor secondary structures, with key parameters that can be adjusted based on the specific organism under investigation (within animals, plants, algae). We illustrate the validity of results from our algorithm using sequencing data for the two Volvocine algae Chlamydomonas reinhardtii (Chlamydomonas) and Volvox carteri (Volvox). Both organisms show little cross-species miRNA sequence conservation, and a heterogeneous miRNA precursor population. We validate our list of Chlamydomonas miRNAs with annotated miRNAs, and demonstrate excellent agreement. Furthermore, we are able to identify additional novel miRNA precursors, with structures ranging from simple mammalian-like hairpins to precursor structures indicating the creation of multiple mature/star miRNA duplexes. Novel miRNAs identified in Volvox show no similarity to mature miRNAs in Chlamydomonas. These results confirm the need for conservationindependent miRNA identification methods. Examination of small RNAs of Volvox carteri during different stages of its life cycle
Project description:We identified 174 miRNAs expressed in Volvox carteri. Some of Volvox miRNAs are highly enriched in gonidia or somatic cells. Subsequently, we predicted the targets of Volvox miRNAs and found many of target genes were regulated through mRNA degradation. Conservation analysis suggests the common origin of miRNA between Volvox and Chlamydomonas and high frequency of birth and death of Volvox miRNAs. Identification of miRNAs in multicellular green Volvox
Project description:Germ-soma differentiation is a hallmark of complex multicellular organisms, yet its origins are not well understood. Volvox carteri is a simple multicellular green alga that has recently evolved a simple germ-soma dichotomy with only two cell types: large germ cells called gonidia and small terminally differentiated somatic cells. Here, we provide a comprehensive characterization of the gonidial and somatic transcriptomes of Volvox to uncover fundamental differences between the molecular and metabolic programming of these cell types. We found extensive transcriptome differentiation between cell types, with somatic cells expressing a more specialized program overrepresented in younger, lineage-specific genes and gonidial cells expressing a more generalist program overrepresented in more ancient genes that shared striking overlap with metazoan stem-cell-specific genes. Directed analyses of metabolic pathways revealed a strong dichotomy between cell types with gonidial cells expressing growth-related genes and somatic cells expressing an altruistic metabolic program geared towards the assembly of flagella, which support organismal motility, and the conversion of storage carbon to sugars, which act as donors of extracellular matrix glycoproteins whose secretion enables massive organismal expansion. Volvox orthologs of Chlamydomonas diurnally regulated genes were analyzed for cell-type distribution and found to be strongly partitioned, with expression of dark-phase genes overrepresented in somatic cells and light-phase genes overrepresented in gonidial cells, a result that is consistent with cell type programs in Volvox arising by cooption of temporal regulons in a unicellular ancestor. Together our findings reveal fundamental molecular, metabolic, and evolutionary mechanisms that underlie the origins of germ-soma differentiation in Volvox and provide a template for understanding the acquisition of germ-soma differentiation in other multicellular lineages. Overall design: Gonidial cells and somatic cells of Volvox were separated and RNA was extracted from each cell type pool. RNA-seq libraries were generated from the extracted RNA and sequenced using high-throughput Illumina sequencing. A total of 4 RNA-seq samples were generated, 2 gonidial cell biological replicates and 2 somatic cell biological replicates.
Project description:RNA populations in Chlamydomonas reinhardtii Keywords: Highly parallel pyrosequencing Small RNAs were prepared from Chlamydomonas reinhardtii total extracts,ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of 454 cloning primers and provided to 454 Life Sciences (Branford, CT) for sequencing. For technical details, see Tao Zhao, Guanglin Li, Shijun Mi, Shan Li, Gregory J. Hannon, Xiu-Jie Wang, and Yijun Qi. 2007. A Complex System of Small RNAs in the Unicellular Green Alga Chlamydomonas reinhardtii. Genes & Development
Project description:The recent identification of catalytically active peptidylglycine -amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). Homologs of prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) were identified in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectroscopy to identify proteins recovered from the soluble secretome of mating gametes and searched for evidence that the putative peptidergic processing enzymes were functional.After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact or were subjected to cleavage were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the soluble mating secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites and had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from cultures of Volvox carteri (in control, phosphate starvation and sulphate starvation conditions). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from cultures of Volvox carteri in control, phosphate starvation and sulphate starvation conditions. Total RNA was isolated using the TriReagent (Molecular Research Center), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Mapping 6mA at single-base resolution across multiple eukaryotic genomes reveals its genomic distribution patterns, indicating a function in transcriptional regulation. Overall design: Using genome-wide methylation detection by single-molecule sequencing, we mapped 6mA at single-base resolution in seven eukaryotic species including the model plant Arabidopsis thaliana, the green alga Chlamydomonas reinhardtii, the marine alga Nannochloropsis oceanica, and the wood-degrading fungus Auricularia delicata. We also performed methylation mapping of publicly available raw SMRT sequencing data from two animal models, Drosophila melanogaster and Caenorhabditis elegans, and budding yeast, Saccharomyces cerevisiae.