Combining Insertion and Deletion in RNA-editing Preserves Regularity

Inspired by RNA-editing as occurs in transcriptional processes in the living cell, we introduce an abstract notion of string adjustment, called guided rewriting. This formalism allows simultaneously inserting and deleting elements. We prove that guided rewriting preserves regularity: for every regular language its closure under guided rewriting is regular too. This contrasts an earlier abstraction of RNA-editing separating insertion and deletion for which it was proved that regularity is not preserved. The particular automaton construction here relies on an auxiliary notion of slice sequence which enables to sweep from left to right through a completed rewrite sequence.Comment: In Proceedings MeCBIC 2012, arXiv:1211.347

Analysis pipelines for cancer genome sequencing in mice

Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2?7 d, depending on the desired analyses.D.S. is supported by the European Research Council (Consolidator Grant 648521) and the Deutsche Forschungsgemeinschaft (SA1374/4-2; SFB 1321). I.V. is supported by the European Research Council (Starting Grant INTRAHETEROSEQ) and the Spanish Goverment (SAF2016-76758-R). R.R. is supported by the European Research Council (Consolidator Grants PACA-MET and MSCA-ITN-ETN PRECODE), the Deutsche Forschungsgemeinschaft (DFG RA1629/2-1; SFB1243; SFB1321; SFB1335), the German Cancer Consortium Joint Funding Program, and the Deutsche Krebshilfe (70112480)

Hidden stitches: RNA cryptic splicing and its role in human disease

A great majority of human genes contain introns: tracts of mostly non-functional sequence that intervene the functional exons. When intron-bearing genes are transcribed into RNA, the introns are removed from the transcript via splicing, a process controlled by a multimolecular assembly called the spliceosome. Although splicing is generally well-regulated, the spliceosome sometimes splices RNA transcripts at sites other than their canonical exon boundaries. This “cryptic” splicing can be a random event, part of an unidentified regulatory process, the effect of a mutation, or the result of other perturbances to the spliceosome’s normal behaviour. In this thesis, I present four reports on the mechanisms underlying certain forms of cryptic splicing. In the first report, an analysis of pathogenic pseudoexons in the DMD gene reveals that each causative mutation falls into a distinct category defined by its proximity to the pseudoexon, and that many DMD pseudoexon splice sites are actively spliced in non-mutant cells. The second report builds on this by constructing a catalogue of over 400 pseudoexon variants from across the human transcriptome and uses this dataset to propose new and revised pseudoexon mutation categories. Like the first report, this second report also finds substantial congruence between pseudoexons and active deep intronic splice sites – including several recursive splice sites – suggesting a causal link between these phenomena. A third report explores how some cryptic exons may provide an explanatory mechanism to connect common genetic variants with their associated population phenotypes and outlines a simple method for discovering new examples. The fourth and final report uses RNA secondary structure modelling to explain why some antisense oligonucleotides can induce partial exon skipping through cryptic splice-site activation. Collectively, these reports present several novel insights into the causes of cryptic splicing and offer suggestions for how future research may build upon these insights

Understanding the dynamic regulation of SOCS3

In the past two decades, it has become evident that signal transduction pathways are more than two dimensional pathways consisted of proteins that are just activated or supressed in response to distinct cues. Instead, the dynamic nature of key proteins regulates the strength and quality of the signal. Several key signal transduction pathways are controlled by negative feedback loops that are highly dynamic and demonstrate oscillatory behaviours. Negative feedback regulation of the JAK/STAT pathway by Suppressors of Cytokine Signalling (SOCS) is an example of oscillatory signalling. We sought to investigate the oscillatory capacity of the tumour suppressor protein SOCS3 and its role in important cellular functions using whole-cell population and single-cell analysis. An important aspect of cell biology using experimental cell-population techniques is to produce a synchronized cell culture. Serum starvation and subsequent shock is able to capture the oscillatory behaviour of SOCS3 protein to some extent. However, the average response in whole-cell population systems demonstrated to be ‘noisy’ leading to establishment of a single-cell analysis system. To investigate SOCS3 oscillation at the single cell level, we first attempted to generate cell clones stably expressing SOCS3 C-terminal GFPSpark fusion protein from its respective endogenous promoter to monitor its expression in real time with confocal microscopy. Despite careful optimization of each step of CRISPR/Cas9 strategy, the generation of GFPSpark knockin cell line was not successful. Finally, we utilised the tandem fluorescent protein timer (tFT) strategy to investigate localisation and trafficking of SOCS3 protein and monitor its promoter activity in response to different stimuli. The use of tFT provided us the ability to analyse SOCS3 dynamics across spatial and temporal dimensions under either normal culture conditions or different treatments that are known to influence on SOCS3 half-life and degradation rates

LIPIcs, Volume 248, ISAAC 2022, Complete Volume

LIPIcs, Volume 248, ISAAC 2022, Complete Volum

A cooperative framework for molecular biology database integration using image object selection

The theme and the concept of 'Molecular Biology Database Integration' and the problems associated with this concept initiated the idea for this Ph.D research. The available technologies facilitate to analyse the data independently and discretely but it fails to integrate the data resources for more meaningful information. This along with the integration issues created the scope for this Ph.D research. The research has reviewed the 'database interoperability' problems and it has suggested a framework for integrating the molecular biology databases. The framework has proposed to develop a cooperative environment to share information on the basis of common purpose for the molecular biology databases. The research has also reviewed other implementation and interoperability issues for laboratory based, dedicated and target specific database. The research has addressed the following issues: diversity of molecular biology databases schemas, schema constructs and schema implementation multi-database query using image object keying, database integration technologies using context graph, automated navigation among these databases. This thesis has introduced a new approach for database implementation. It has introduced an interoperable component database concept to initiate multidatabase query on gene mutation data. A number of data models have been proposed for gene mutation data which is the basis for integrating the target specific component database to be integrated with the federated information system. The proposed data models are: data models for genetic trait analysis, classification of gene mutation data, pathological lesion data and laboratory data. The main feature of this component database is non-overlapping attributes and it will follow non-redundant integration approach as explained in the thesis. This will be achieved by storing attributes which will not have the union or intersection of any attributes that exist in public domain molecular biology databases. Unlike data warehousing technique, this feature is quite unique and novel. The component database will be integrated with other biological data sources for sharing information in a cooperative environment. This involves developing new tools. The thesis explains the role of these new tools which are: meta data extractor, mapping linker, query generator and result interpreter. These tools are used for a transparent integration without creating any global schema of the participating databases. The thesis has also established the concept of image object keying for multidatabase query and it has proposed a relevant algorithm for matching protein spot in gel electrophoresis image. An object spot in gel electrophoresis image will initiate the query when it is selected by the user. It matches the selected spot with other similar spots in other resource databases. This image object keying method is an alternative to conventional multidatabase query which requires writing complex SQL scripts. This method also resolve the semantic conflicts that exist among molecular biology databases. The research has proposed a new framework based on the context of the web data for interactions with different biological data resources. A formal description of the resource context is described in the thesis. The implementation of the context into Resource Document Framework (RDF) will be able to increase the interoperability by providing the description of the resources and the navigation plan for accessing the web based databases. A higher level construct is developed (has, provide and access) to implement the context into RDF for web interactions. The interactions within the resources are achieved by utilising an integration domain to extract the required information with a single instance and without writing any query scripts. The integration domain allows to navigate and to execute the query plan within the resource databases. An extractor module collects elements from different target webs and unify them as a whole object in a single page. The proposed framework is tested to find specific information e.g., information on Alzheimer's disease, from public domain biology resources, such as, Protein Data Bank, Genome Data Bank, Online Mendalian Inheritance in Man and local database. Finally, the thesis proposes further propositions and plans for future work

Evaluation of a precision medicine approach for hnRNP U-related developmental epileptic encephalopathy using a mouse model of disease

Mutations in genes that cause transcriptional dysregulation, such as genes that encode DNA and RNA-binding proteins (RNABPs), are a well-described cause of neurodevelopmental syndromes such as autism and epilepsy. Heterozygous de novo mutations involving the gene HNRNPU, which encodes the heterogeneous nuclear ribonuclear protein U, have been implicated in a neurodevelopmental syndrome most commonly characterized by epileptic encephalopathy. Although hnRNP U is a highly-abundant and ubiquitously-expressed DNA- and RNA-binding protein involved in a variety of important nuclear processes—most notably gene expression regulation—the role it plays in neurological disease is unclear and has yet to be studied. The work presented here examines a precision medicine approach for epilepsies thought to have a transcriptomic basis, starting with a thorough neurophysiological characterization of a heterozygous loss-of-function Hnrnpu mouse model (Hnrnpu+/113DEL), followed by a comprehensive and region-specific single-cell transcriptomic study, and finally the validation of implicated brain regions. Characterization of the Hnrnpu+/113DEL mouse line revealed an increased susceptibility to seizures in Hnrnpu+/113DEL mice, along with an increased perinatal mortality, global developmental delay and gait abnormalities. Gene expression profiling, including bulk RNA-sequencing of neocortex and single cell RNA-sequencing of both neocortex and hippocampus, revealed widespread, yet modest, dysregulation of gene expression that was largely inversely correlated to gene-length, and involved important, neurodevelopmental disease genes. In particular, pyramidal neurons of the subiculum displayed greater transcriptional burden upon heterozygous loss of Hnrnpu, with the known epilepsy gene Mef2c as a clear outlier showing greater than 50% reduction in expression. Follow-up investigation into whether this region- and cell-type specific gene dysregulation correlated to differences in neuronal function using c-Fos immunostaining, revealed an overall decrease in neuronal activity within the ventral subiculum in Hnrnpu+/113DEL mice. In summary, our data validates the presence of neurodevelopmental defects upon heterozygous loss of Hnrnpu and supports the notion of transcriptional dysregulation as a likely contributing factor to hnRNP U-related disease, possibly through the dysfunction of subiculum-derived excitatory neurons. Future studies evaluating the relationship between reduced activity within the ventral subiculum and hnRNP U disease phenotypes are an important next step, and may serve as the basis for targeted therapeutic discovery

Abstracts from the 50th European Society of Human Genetics Conference: Posters

International audienc

Advances in Molecular Breeding of Vegetable Crops

Vegetable crops provide valuable minerals and vitamins that are indispensible for human health. Scientists have been working on the genetics of vegetable crops, deciphering the molecular bases of agronomically important traits. These genetic bases and variations in vegetable traits will greatly facilitate vegetable genetic improvement. Therefore, the genes of and genetic research on vegetable crops are of great importance. This Special Issue is a collection of 13 important research papers addressing the genes, genetics, and breeding of major vegetable crops. In the present book, the authors described the genes and QTLs responsible for stress tolerance, disease resistance, vegetable yield, and quality. The 13 research papers cover germplasm enhancement and evaluation, QTL mapping, gene isolation, marker development, and gene expression as well as gene editing in a wide range of vegetable species, including broccoli, pepper, eggplant, onion, and Cucurbita species. Readers from all over the globe are expected to greatly benefit from this Special Issue collection regarding their own work and the goal of improving breeding efficiency with molecular breeding to generate environment-adaptive, high-yield, and high-quality vegetable crops with which to feed the global population of 9.7 billion in an extreme climate by 2050