@phdthesis{Childs2010, author = {Childs, Liam H.}, title = {Bioinformatics approaches to analysing RNA mediated regulation of gene expression}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-41284}, school = {Universit{\"a}t Potsdam}, year = {2010}, abstract = {The genome can be considered the blueprint for an organism. Composed of DNA, it harbours all organism-specific instructions for the synthesis of all structural components and their associated functions. The role of carriers of actual molecular structure and functions was believed to be exclusively assumed by proteins encoded in particular segments of the genome, the genes. In the process of converting the information stored genes into functional proteins, RNA - a third major molecule class - was discovered early on to act a messenger by copying the genomic information and relaying it to the protein-synthesizing machinery. Furthermore, RNA molecules were identified to assist in the assembly of amino acids into native proteins. For a long time, these - rather passive - roles were thought to be the sole purpose of RNA. However, in recent years, new discoveries have led to a radical revision of this view. First, RNA molecules with catalytic functions - thought to be the exclusive domain of proteins - were discovered. Then, scientists realized that much more of the genomic sequence is transcribed into RNA molecules than there are proteins in cells begging the question what the function of all these molecules are. Furthermore, very short and altogether new types of RNA molecules seemingly playing a critical role in orchestrating cellular processes were discovered. Thus, RNA has become a central research topic in molecular biology, even to the extent that some researcher dub cells as "RNA machines". This thesis aims to contribute towards our understanding of RNA-related phenomena by applying Bioinformatics means. First, we performed a genome-wide screen to identify sites at which the chemical composition of DNA (the genotype) critically influences phenotypic traits (the phenotype) of the model plant Arabidopsis thaliana. Whole genome hybridisation arrays were used and an informatics strategy developed, to identify polymorphic sites from hybridisation to genomic DNA. Following this approach, not only were genotype-phenotype associations discovered across the entire Arabidopsis genome, but also regions not currently known to encode proteins, thus representing candidate sites for novel RNA functional molecules. By statistically associating them with phenotypic traits, clues as to their particular functions were obtained. Furthermore, these candidate regions were subjected to a novel RNA-function classification prediction method developed as part of this thesis. While determining the chemical structure (the sequence) of candidate RNA molecules is relatively straightforward, the elucidation of its structure-function relationship is much more challenging. Towards this end, we devised and implemented a novel algorithmic approach to predict the structural and, thereby, functional class of RNA molecules. In this algorithm, the concept of treating RNA molecule structures as graphs was introduced. We demonstrate that this abstraction of the actual structure leads to meaningful results that may greatly assist in the characterization of novel RNA molecules. Furthermore, by using graph-theoretic properties as descriptors of structure, we indentified particular structural features of RNA molecules that may determine their function, thus providing new insights into the structure-function relationships of RNA. The method (termed Grapple) has been made available to the scientific community as a web-based service. RNA has taken centre stage in molecular biology research and novel discoveries can be expected to further solidify the central role of RNA in the origin and support of life on earth. As illustrated by this thesis, Bioinformatics methods will continue to play an essential role in these discoveries.}, language = {en} } @article{BerryRosaHowardetal.2017, author = {Berry, Scott and Rosa, Stefanie and Howard, Martin and Buhler, Marc and Dean, Caroline}, title = {Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression}, series = {Genes \& Development}, volume = {31}, journal = {Genes \& Development}, publisher = {Cold Spring Harbor Laboratory Press}, address = {Cold Spring Harbor, NY}, issn = {0890-9369}, doi = {10.1101/gad.305227.117}, pages = {2115 -- 2120}, year = {2017}, abstract = {Epigenetic maintenance of gene repression is essential for development. Polycomb complexes are central to this memory, but many aspects of the underlying mechanism remain unclear. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binds Polycomb-deposited H3K27me3 and is required for repression of many Polycomb target genes in Arabidopsis. Here we show that LHP1 binds RNA in vitro through the intrinsically disordered hinge region. By independently perturbing the RNA-binding hinge region and H3K27me3 (trimethylation of histone H3 at Lys27) recognition, we found that both facilitate LHP1 localization and H3K27me3 maintenance. Disruption of the RNAbinding hinge region also prevented formation of subnuclear foci, structures potentially important for epigenetic repression.}, language = {en} } @article{LombardoOttenAbdelilahSeyfried2015, author = {Lombardo, Veronica A. and Otten, Cecile and Abdelilah-Seyfried, Salim}, title = {Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis}, series = {Journal of visualized experiments}, journal = {Journal of visualized experiments}, number = {95}, publisher = {JoVE}, address = {Cambridge}, issn = {1940-087X}, doi = {10.3791/52087}, pages = {7}, year = {2015}, abstract = {The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.}, language = {en} } @misc{LukanMachensColletal.2018, author = {Lukan, Tjaša and Machens, Fabian and Coll, Anna and Baebler, Špela and Messerschmidt, Katrin and Gruden, Kristina}, title = {Plant X-tender}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {990}, issn = {1866-8372}, doi = {10.25932/publishup-44628}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-446281}, pages = {21}, year = {2018}, abstract = {Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scarfree and sequence-independent multigene assembly strategy AssemblX,based on overlapdepended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.}, language = {en} } @article{SchudomaLarhlimiWalther2011, author = {Schudoma, Christian and Larhlimi, Abdelhalim and Walther, Dirk}, title = {The influence of the local sequence environment on RNA loop structures}, series = {RNA : a publication of the RNA Society}, volume = {17}, journal = {RNA : a publication of the RNA Society}, number = {7}, publisher = {Cold Spring Harbor Laboratory Press}, address = {Cold Spring Harbor, NY}, issn = {1355-8382}, doi = {10.1261/rna.2550211}, pages = {1247 -- 1257}, year = {2011}, abstract = {RNA folding is assumed to be a hierarchical process. The secondary structure of an RNA molecule, signified by base-pairing and stacking interactions between the paired bases, is formed first. Subsequently, the RNA molecule adopts an energetically favorable three-dimensional conformation in the structural space determined mainly by the rotational degrees of freedom associated with the backbone of regions of unpaired nucleotides (loops). To what extent the backbone conformation of RNA loops also results from interactions within the local sequence context or rather follows global optimization constraints alone has not been addressed yet. Because the majority of base stacking interactions are exerted locally, a critical influence of local sequence on local structure appears plausible. Thus, local loop structure ought to be predictable, at least in part, from the local sequence context alone. To test this hypothesis, we used Random Forests on a nonredundant data set of unpaired nucleotides extracted from 97 X-ray structures from the Protein Data Bank (PDB) to predict discrete backbone angle conformations given by the discretized eta/theta-pseudo-torsional space. Predictions on balanced sets with four to six conformational classes using local sequence information yielded average accuracies of up to 55\%, thus significantly better than expected by chance (17\%-25\%). Bases close to the central nucleotide appear to be most tightly linked to its conformation. Our results suggest that RNA loop structure does not only depend on long-range base-pairing interactions; instead, it appears that local sequence context exerts a significant influence on the formation of the local loop structure.}, language = {en} }