Selective Translation of Low Abundance and Upregulated Transcripts in Halobacterium salinarum.

When organisms encounter an unfavorable environment, they transition to a physiologically distinct, quiescent state wherein abundant transcripts from the previous active growth state continue to persist, albeit their active transcription is downregulated. In order to generate proteins for the new quiescent physiological state, we hypothesized that the translation machinery must selectively translate upregulated transcripts in an intracellular milieu crowded with considerably higher abundance transcripts from the previous active growth state. Here, we have analyzed genome-wide changes in the transcriptome (RNA sequencing [RNA-seq]), changes in translational regulation and efficiency by ribosome profiling across all transcripts (ribosome profiling [Ribo-seq]), and protein level changes in assembled ribosomal proteins (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) to investigate the interplay of transcriptional and translational regulation in Halobacterium salinarum as it transitions from active growth to quiescence. We have discovered that interplay of regulatory processes at different levels of information processing generates condition-specific ribosomal complexes to translate preferentially pools of low abundance and upregulated transcripts. Through analysis of the gene regulatory network architecture of H. salinarum, Escherichia coli, and Saccharomyces cerevisiae, we demonstrate that this conditional, modular organization of regulatory programs governing translational systems is a generalized feature across all domains of life.IMPORTANCE Our findings demonstrate conclusively that low abundance and upregulated transcripts are preferentially translated, potentially by environment-specific translation systems with distinct ribosomal protein composition. We show that a complex interplay of transcriptional and posttranscriptional regulation underlies the conditional and modular regulatory programs that generate ribosomes of distinct protein composition. The modular regulation of ribosomal proteins with other transcription, translation, and metabolic genes is generalizable to bacterial and eukaryotic microbes. These findings are relevant to how microorganisms adapt to unfavorable environments when they transition from active growth to quiescence by generating proteins from upregulated transcripts that are in considerably lower abundance relative to transcripts associated with the previous physiological state. Selective translation of transcripts by distinct ribosomes could form the basis for adaptive evolution to new environments through a modular regulation of the translational systems

Patterns and Complexity in Biological Systems: A Study of Sequence Structure and Ontology-based Networks

Biological information can be explored at many different levels, with the most basic information encoded in patterns within the DNA sequence. Through molecular level processes, these patterns are capable of controlling the states of genes, resulting in a complex network of interactions between genes. Key features of biological systems can be determined by evaluating properties of this gene regulatory network. More specifically, a network-based approach helps us to understand how the collective behavior of genes corresponds to patterns in genetic function. We combine Chromatin-Immunoprecipitation microarray (ChIP-chip) data with genomic sequence data to determine how DNA sequence works to recruit various proteins. We quantify this information using a value termed "nmer-association.'' "Nmer-association'' measures how strongly individual DNA sequences are associated with a protein in a given ChIP-chip experiment. We also develop the "split-motif'' algorithm to study the underlying structural properties of DNA sequence independent of wet-lab data. The "split-motif'' algorithm finds pairs of DNA motifs which preferentially localize relative to one another. These pairs are primarily composed of known transcription factor binding sites and their co-occurrence is indicative of higher-order structure. This kind of structure has largely been missed in standard motif-finding algorithms despite emerging evidence of the importance of complex regulation. In both simple and complex regulation, two genes that are connected in a regulatory fashion are likely to have shared functions. The Gene Ontology (GO) provides biologists with a controlled terminology with which to describe how genes are associated with function and how those functional terms are related to each other. We introduce a method for processing functional information in GO to produce a gene network. We find that the edges in this network are correlated with known regulatory interactions and that the strength of the functional relationship between two genes can be used as an indicator of how informationally important that link is in the regulatory network. We also investigate the network structure of gene-term annotations found in GO and use these associations to establish an alternate natural way to group the functional terms. These groups of terms are drastically different from the hierarchical structure established by the Gene Ontology and provide an alternative framework with which to describe and predict the functions of experimentally identified groups of genes

Analysis of DNA-binding Proteins in Yeast Saccharomyces Cerevisiae

Gene expression is an elaborate and finely tuned process involving the regulated interactions of multiple proteins with promoter and enhancer elements. A variety of approaches are currently used to study these interactions in vivo, in vitro as well as in silico. With the genome sequences of many organisms now readily available, a plethora of DNA functional elements have been predicted, but the process of identifying the proteins that bind to them in vivo remains a bottleneck. I developed two high-throughput assays to address this issue. The first is a modification of the yeast one-hybrid assay. The second is probing protein microarrays with DNA sequence elements. Using these methods, I identified two proteins, Sef1 and Yjl103c, that bind to the same DNA sequence element. Sef1 and Yjl103c are little-characterized members of the zinc cluster family of transcription factors of S. cerevisiae. Characterization of their mechanism of action as well as identification of some of their target genes leads to the conclusion that they play a pivotal role in the transcriptional regulation of utilization of nonfermentable carbon sources by budding yeast

Knowledge derivation and data mining strategies for probabilistic functional integrated networks

PhDOne of the fundamental goals of systems biology is the experimental verification of the interactome: the entire complement of molecular interactions occurring in the cell. Vast amounts of high-throughput data have been produced to aid this effort. However these data are incomplete and contain high levels of both false positives and false negatives. In order to combat these limitations in data quality, computational techniques have been developed to evaluate the datasets and integrate them in a systematic fashion using graph theory. The result is an integrated network which can be analysed using a variety of network analysis techniques to draw new inferences about biological questions and to guide laboratory experiments. Individual research groups are interested in specific biological problems and, consequently, network analyses are normally performed with regard to a specific question. However, the majority of existing data integration techniques are global and do not focus on specific areas of biology. Currently this issue is addressed by using known annotation data (such as that from the Gene Ontology) to produce process-specific subnetworks. However, this approach discards useful information and is of limited use in poorly annotated areas of the interactome. Therefore, there is a need for network integration techniques that produce process-specific networks without loss of data. The work described here addresses this requirement by extending one of the most powerful integration techniques, probabilistic functional integrated networks (PFINs), to incorporate a concept of biological relevance. Initially, the available functional data for the baker’s yeast Saccharomyces cerevisiae was evaluated to identify areas of bias and specificity which could be exploited during network integration. This information was used to develop an integration technique which emphasises interactions relevant to specific biological questions, using yeast ageing as an exemplar. The integration method improves performance during network-based protein functional prediction in relation to this process. Further, the process-relevant networks complement classical network integration techniques and significantly improve network analysis in a wide range of biological processes. The method developed has been used to produce novel predictions for 505 Gene Ontology biological processes. Of these predictions 41,610 are consistent with existing computational annotations, and 906 are consistent with known expert-curated annotations. The approach significantly reduces the hypothesis space for experimental validation of genes hypothesised to be involved in the oxidative stress response. Therefore, incorporation of biological relevance into network integration can significantly improve network analysis with regard to individual biological questions

Synthesis, characterization, and self-assembly of porphyrins conjugated to superparamagnetic colloidal particles for enhanced photodynamic therapy

Porphyrins and their derivatives are often used as photosensitizers in photodynamic therapy (PDT), which is a noninvasive antitumor treatment. The photochemical process for PDT involves exciting a photosensitizing agent with visible light, which induces cytotoxicity in the presence of oxygen as a result of forming reactive oxygen species (ROS). The ROS are the responsible components for invoking cell death and destruction of tumors. Although this mechanism is an effective cancer therapeutic, it still has many shortcomings. One major challenge of PDT concerns improving the tumor selectivity and specificity of photosensitizers because porphyrins have nonspecific affinity to tumor cells. The discussed research introduces a potential drug delivery vehicle to enhance the efficacy of cancer therapeutics and overcome the aforementioned issues. Specifically, hybrid composite particles composed of superparamagnetic polypeptide-coated silica nanoparticles conjugated to porphyrins were designed to improve the mechanism of tumor cell destruction via controlled assembly and transport. Along the path of developing these nanocomposites, porphyrin self-assembly was explored to understand the dynamics of porphyrins alone. A series of complementary experiments and analytical methods were used, including UV-Vis and fluorescence spectroscopy measurements, small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and freeze-fracture transmission electron microscopy (FF-TEM). Whereas UV-Vis and fluorescence techniques enabled us to determine the type of aggregates formed, AUC and SAXS provided complementary details and information about the size of the assemblies in solution. Cryo-TEM and FF-TEM provided direct visualization of the aggregates

Organisational and signalling cues in membranes

Recently, a new class of membrane-shapers termed N-Ank proteins (Wolf et al., 2019) were defined by their ability to bind, sense, and shape the membrane via their N-Ank module, which consists of an amino(N)-terminal amphipathic helix and ankyrin repeats. N-Ank family of membrane-shapers is phylogenetically classified into two sub-families and Ankyrin repeat domain-containing protein (Ankrd) 24, an uncharacterised protein, was suggested to be in their smaller sub-family. Thus, this study unveiled that Ankrd24 isoform 6 (Ankrd24x6) is one of the expressed isoforms in brains of 8-week-old mice. Investigation of the N-Ank properties of Ankrd24x6 using in vitro reconstitution assays with liposomes showed that it is an N-Ank protein but with distinct characteristics. The N-Ank module of Ankrd24x6 binds and shape the membrane and displayed that the presence of the putative amphipathic helix is quintessential for its binding. The predicted seven ankyrin repeats in Ankrd24x6 displayed lack of binding to the liposomes irrespective of their curvature. This property of Ankrd24x6 is distinct compared to the other members of the N-Ank superfamily which showed that the ankyrin repeats alone preferred higher curvature liposomes. Ankrd24x6 is also capable of oligomerising as it demonstrated self association, which was mediated by the predicted coiled coil domains. Another interesting feature of Ankrd24x6 is the presence of a proline-rich motif via which it showed binding to the syndapin I protein. Syndapin I is involved in diverse crucial biological processes like synaptic vesicle recycling, cytoskeletal remodelling, dendritogenesis and ciliogenesis because it is capable of binding and shaping the membranes and can additionally interact with various proteins. ..

Functional characterisation of the mitochondrial Hsp70 cochaperone Zim17

The mitochondrial zinc finger protein Zim17 belongs to a newly identified class of cochaperones that maintain the function of Hsp70 proteins in mitochondria and plastides of eukaryotic cells, presumably by preventing the aggregation of their respective chaperone partners. However, while its aggregation-preventive function is well demonstrated in vitro, little is known about the influence of the Zim17 interaction on the chaperone activity of mitochondrial Hsp70s (mtHsp70s) and its concurrent effects in the cellular context. Due to the aggregation-protective character of Zim17, recombinant co-expression with the zinc finger protein allowed the purification of the main yeast mtHsp70 Ssc1 from E.coli cells under native conditions. The purified proteins were used to analyse the influence of Zim17 on the solubility of Ssc1 as well as the character and stability of its binding to the chaperone. Zim17 interacted with Ssc1 as a single molecule but tended to form dimers in the absence of the Hsp70 chaperone. Though the presence of Zim17 improved the solubility of recombinant Ssc1 in E.coli cells, substantial amounts of the Hsp70 chaperone still aggregated, even when Zim17 was expressed in saturated amounts. To study the effects of a loss of Zim17 function in the cellular environment, novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae were generated. Yeast cells carrying these mutations showed a temperature- sensitive growth phenotype and a tendency to develop respiratory deficits. On fermentable growth media, the mutant cells were prone to loose their respiratory competence and were inviable at elevated temperatures. In these cells, a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis was observed. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. The induction of the zim17 mutant phenotype by subjection to a high temperature treatment lead to a strong import defect for Ssc1-dependent matrix-targeted precursor proteins that correlated with a significantly reduced binding of newly imported substrate proteins to Ssc1. Both in vitro and in vivo approaches point to the conclusion that Zim17 is not primarily required for the maintenance of mtHsp70 solubility. Instead, a functional analysis of the chaperone cycles of Ssc1 and Ssq1 shows that Zim17 directly assists the functional interaction of mtHsp70 with substrate proteins in a J-protein cochaperone-dependent manner

Transcriptome Analysis of MRG-1-deficient Caenorhabditis elegans animals using short and long read sequencing

Das Schicksal einer differenzierten Zelle wird durch epigenetische Grenzen bestimmt und mittels Schutzmechanismen bewahrt, wodurch die Reprogrammierung in andere Zelltypen verhindert wird. In dieser Studie haben wir ein Chromatin-regulierendes Protein, das konservierte MORF4-Verwandte-Gen (MRG) Protein MRG-1, als Barriere für die Reprogrammierung von Zellen in Caenorhabditis elegans (C. elegans) identifiziert. RNAi gegen MRG-1 ermöglicht es uns Keimzellen mittels Überexpression des Neuronen-induzierenden Transkriptionsfaktors CHE-1 in neuronenartige Zellen umzuwandeln. Mittels ChIP-seq fanden wir heraus, dass MRG-1 unterschiedliche DNA Bindungsstellen in den Keimbahnen und somatischen Geweben von C. elegans aufweist. Wir konnten zeigen, dass MRG-1 besonders stark am Genkörper angereichert ist und sich hauptsächlich auf Genen befindet, welche die aktive Histonmarkierung H3K36me3 tragen. Die Charakterisierung der Protein-Protein-Interaktionspartner von MRG-1 mittels Co-IP/MS ergab, dass MRG-1 mit der Histon-H3K9-Methyltransferase SET-26 und der b-gebundenen N-Acetylglucosamin Transferase OGT-1 zusammenarbeitet, um die Umwandlung von Keimzellen in Neuronen zu verhindern. Basierend auf RNA-Seq Experimenten in mrg-1-Mutanten und Wildtyp konnten wir weitreichende Veränderungen der Genexpression mit Auswirkung auf Signalwege wie den Notch Signalweg enthüllen, welcher bekanntermaßen die Zelltyp-Reprogrammierung fördern. Mittels Long-Read basiertem RNA-seq in mrg-1-Mutanten und der Integration entsprechender ChIP-seq Daten habe ich die Beteiligung von MRG-1 am prä-mRNA-Spleißen in C. elegans gezeigt, analog zum Säugetierortholog MRG15. Diese Ergebnisse weisen darauf hin, dass MRG-1 durch die Regulierung des Chromatins und die Sicherstellung des korrekten Spleißens die Expressionsniveaus kritischer Gene und Signalwege aufrechterhält, um eine ordnungsgemäße Keimbahnentwicklung zu gewährleisten und das Schicksal der Keimzellen zu schützen.Das Schicksal einer differenzierten Zelle wird durch epigenetische Grenzen bestimmt und mittels Schutzmechanismen bewahrt, wodurch die Reprogrammierung in andere Zelltypen verhindert wird. In dieser Studie haben wir ein Chromatin-regulierendes Protein, das konservierte MORF4-Verwandte-Gen (MRG) Protein MRG-1, als Barriere für die Reprogrammierung von Zellen in Caenorhabditis elegans (C. elegans) identifiziert. RNAi gegen MRG-1 ermöglicht es uns Keimzellen mittels Überexpression des Neuronen-induzierenden Transkriptionsfaktors CHE-1 in neuronenartige Zellen umzuwandeln. Mittels ChIP-seq fanden wir heraus, dass MRG-1 unterschiedliche DNA Bindungsstellen in den Keimbahnen und somatischen Geweben von C. elegans aufweist. Wir konnten zeigen, dass MRG-1 besonders stark am Genkörper angereichert ist und sich hauptsächlich auf Genen befindet, welche die aktive Histonmarkierung H3K36me3 tragen. Die Charakterisierung der Protein-Protein-Interaktionspartner von MRG-1 mittels Co-IP/MS ergab, dass MRG-1 mit der Histon-H3K9-Methyltransferase SET-26 und der b-gebundenen N-Acetylglucosamin Transferase OGT-1 zusammenarbeitet, um die Umwandlung von Keimzellen in Neuronen zu verhindern. Basierend auf RNA-Seq Experimenten in mrg-1-Mutanten und Wildtyp konnten wir weitreichende Veränderungen der Genexpression mit Auswirkung auf Signalwege wie den Notch Signalweg enthüllen, welcher bekanntermaßen die Zelltyp-Reprogrammierung fördern. Mittels Long-Read basiertem RNA-seq in mrg-1-Mutanten und der Integration entsprechender ChIP-seq Daten habe ich die Beteiligung von MRG-1 am prä-mRNA-Spleißen in C. elegans gezeigt, analog zum Säugetierortholog MRG15. Diese Ergebnisse weisen darauf hin, dass MRG-1 durch die Regulierung des Chromatins und die Sicherstellung des korrekten Spleißens die Expressionsniveaus kritischer Gene und Signalwege aufrechterhält, um eine ordnungsgemäße Keimbahnentwicklung zu gewährleisten und das Schicksal der Keimzellen zu schützen