446 research outputs found

    Shedding light on the role of plant miRNAs in DNA damage response (DDR) and trans-kingdom transfer

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    One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential.One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential

    Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain.

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    H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species

    Amphioxus SYCP1 : a case of retrogene replacement and co-option of regulatory elements adjacent to the ParaHox cluster

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    MGG was supported by the University of St Andrews School of Biology Biotechnology and Biological Sciences Research Council DTG and the Wellcome Trust ISSF. Work in the authors’ laboratory is also supported by the Leverhulme Trust.Retrogenes are formed when an mRNA is reverse transcribed and re-inserted into the genome in a location unrelated to the original locus. If this retrocopy inserts into a transcriptionally favourable locus and is able to carry out its original function, it can, in rare cases, lead to retrogene replacement. This involves the original, often multi-exonic, parental copy being lost whilst the newer single-exon retrogene copy ‘replaces’ the role of the ancestral parent gene. One example of this is amphioxus SYCP1, a gene that encodes a protein used in synaptonemal complex formation during meiosis, and which offers the opportunity to examine how a retrogene evolves after the retrogene replacement event. SYCP1 genes exist as large multi-exonic genes in most animals. AmphiSYCP1, however, contains a single coding exon of ~3200bp and has inserted next to the ParaHox cluster of amphioxus, whilst the multi-exonic ancestral parental copy has been lost. Here, we show that AmphiSYCP1 has not only replaced its parental copy, but has evolved additional regulatory function by co- opting a bidirectional promoter from the nearby AmphiCHIC gene. AmphiSYCP1 has also evolved a de novo, multi-exonic 5’untranslated region that displays distinct regulatory states, in the form of two different isoforms, and has evolved novel expression patterns during amphioxus embryogenesis in addition to its ancestral role in meiosis. Absence of ParaHox-like expression of AmphiSYCP1, despite its proximity to the ParaHox cluster, also suggests this gene is not influenced by any potential pan-cluster regulatory mechanisms, which are seemingly restricted to only the ParaHox genes themselves.Publisher PDFPeer reviewe

    Gene expression data analysis using novel methods: Predicting time delayed correlations and evolutionarily conserved functional modules

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    Microarray technology enables the study of gene expression on a large scale. One of the main challenges has been to devise methods to cluster genes that share similar expression profiles. In gene expression time courses, a particular gene may encode transcription factor and thus controlling several genes downstream; in this case, the gene expression profiles may be staggered, indicating a time-delayed response in transcription of the later genes. The standard clustering algorithms consider gene expression profiles in a global way, thus often ignoring such local time-delayed correlations. We have developed novel methods to capture time-delayed correlations between expression profiles: (1) A method using dynamic programming and (2) CLARITY, an algorithm that uses a local shape based similarity measure to predict time-delayed correlations and local correlations. We used CLARITY on a dataset describing the change in gene expression during the mitotic cell cycle in Saccharomyces cerevisiae. The obtained clusters were significantly enriched with genes that share similar functions, reflecting the fact that genes with a similar function are often co-regulated and thus co-expressed. Time-shifted as well as local correlations could also be predicted using CLARITY. In datasets, where the expression profiles of independent experiments are compared, the standard clustering algorithms often cluster according to all conditions, considering all genes. This increases the background noise and can lead to the missing of genes that change the expression only under particular conditions. We have employed a genetic algorithm based module predictor that is capable to identify group of genes that change their expression only in a subset of conditions. With the aim of supplementing the Ustilago maydis genome annotation, we have used the module prediction algorithm on various independent datasets from Ustilago maydis. The predicted modules were cross-referenced in various Saccharomyces cerevisiae datasets to check its evolutionarily conservation between these two organisms. The key contributions of this thesis are novel methods that explore biological information from DNA microarray data

    Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

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    The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type

    Gene expression data analysis using novel methods: Predicting time delayed correlations and evolutionarily conserved functional modules

    Get PDF
    Microarray technology enables the study of gene expression on a large scale. One of the main challenges has been to devise methods to cluster genes that share similar expression profiles. In gene expression time courses, a particular gene may encode transcription factor and thus controlling several genes downstream; in this case, the gene expression profiles may be staggered, indicating a time-delayed response in transcription of the later genes. The standard clustering algorithms consider gene expression profiles in a global way, thus often ignoring such local time-delayed correlations. We have developed novel methods to capture time-delayed correlations between expression profiles: (1) A method using dynamic programming and (2) CLARITY, an algorithm that uses a local shape based similarity measure to predict time-delayed correlations and local correlations. We used CLARITY on a dataset describing the change in gene expression during the mitotic cell cycle in Saccharomyces cerevisiae. The obtained clusters were significantly enriched with genes that share similar functions, reflecting the fact that genes with a similar function are often co-regulated and thus co-expressed. Time-shifted as well as local correlations could also be predicted using CLARITY. In datasets, where the expression profiles of independent experiments are compared, the standard clustering algorithms often cluster according to all conditions, considering all genes. This increases the background noise and can lead to the missing of genes that change the expression only under particular conditions. We have employed a genetic algorithm based module predictor that is capable to identify group of genes that change their expression only in a subset of conditions. With the aim of supplementing the Ustilago maydis genome annotation, we have used the module prediction algorithm on various independent datasets from Ustilago maydis. The predicted modules were cross-referenced in various Saccharomyces cerevisiae datasets to check its evolutionarily conservation between these two organisms. The key contributions of this thesis are novel methods that explore biological information from DNA microarray data

    Kinetoplastid Phylogenomics and Evolution

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    This Special Issue, Kinetoplastid Phylogenomics and Evolution, unites a series of research and review papers related to kinetoplastid parasites. The diverse topics represented in this collection display a variety of scientific questions and methodological approaches currently used to study these fascinating organisms

    Molecular Basis of Apomixis in Plants

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    Apomixis is the consequence of a concerted mechanism that harnesses the sexual machinery and coordinates developmental steps in the ovule to produce an asexual (clonal) seed. Altered sexual developments involve widely characterized functional and anatomical changes in meiosis, gametogenesis, and embryo and endosperm formation. The ovules of apomictic plants skip meiosis and form unreduced female gametophytes whose egg cells develop into a parthenogenetic embryo, and the central cells may or may not fuse to a sperm to develop the seed endosperm. Thus, functional apomixis involves at least three components, apomeiosis, parthenogenesis, and endosperm development, modified from sexual reproduction that must be coordinated at the molecular level to progress through the developmental steps and form a clonal seed. Despite recent progress uncovering specific genes related to apomixis-like phenotypes and the formation of clonal seeds, the molecular basis and regulatorynetwork of apomixis is still unknown. This is a central problem underlying the current limitations of apomixis breeding. This book collates twelve publications addressing different topics around the molecular basis of apomixis, illustrating recent discoveries and advances toward understanding the genetic regulation of the trait, discussing the possible origins of apomixis and the remaining challenges for its commercial deployment in plants
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