Genes2014, 5(4), 1032-1049; doi:10.3390/genes5041032 - published 11 November 2014 Show/Hide Abstract
Abstract: The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.
Abstract: Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.
Abstract: Over the past five years, next generation sequencing has revolutionised the discovery of genes responsible for rare inherited diseases previously resistant to traditional discovery techniques. This review considers how this new technology is being introduced into clinical practice to aid diagnosis and improve the clinical management of individuals and families affected by rare diseases where access to genetic testing was previously limited. We compare and contrast the different approaches that have been adopted including panel based tests, exome and genome sequencing. We provide insights from our own clinical practice demonstrating the challenges and benefits of this new technology.
Abstract: In order to discover novel proteins that promote the nuclear export of newly synthesized mRNAs in mammalian cells, we carried out a limited RNAi screen for proteins required for the proper cytoplasmic distribution of a model intronless mRNA. From this screen we obtained two hits, Ubc9 (SUMO-conjugating E2 enzyme) and GANP (germinal center-associated nuclear protein). Depletion of Ubc9 inhibited the proper cytoplasmic distribution of certain overexpressed intronless mRNAs, while depletion of GANP affected all tested mRNAs. Depletion of Sae1, which is also required for sumoylation, partially inhibited the cytoplasmic distribution of our model mRNA. Interestingly, the block in cytoplasmic accumulation in Ubc9-depleted cells could be overcome if an intron was incorporated into the mRNA. Surprisingly, Ubc9-depleted cells had normal nuclear export of newly synthesized intronless mRNAs, indicating that the observed accumulation of the model mRNA in the nuclei of transfected cells was likely due to some more general perturbation. Indeed, depletion of Ubc9, coupled with the overexpression of the intronless mRNAs, caused the redistribution of the nuclear speckle protein SC35 to cytoplasmic foci. Our results suggest that sumoylation may play a role in the proper assembly of mRNPs and/or the distribution of key RNA binding proteins, and may thus contribute to general protein expression patterns.
Abstract: The High-performance Integrated Virtual Environment (HIVE) is a high-throughput cloud-based infrastructure developed for the storage and analysis of genomic and associated biological data. HIVE consists of a web-accessible interface for authorized users to deposit, retrieve, share, annotate, compute and visualize Next-generation Sequencing (NGS) data in a scalable and highly efficient fashion. The platform contains a distributed storage library and a distributed computational powerhouse linked seamlessly. Resources available through the interface include algorithms, tools and applications developed exclusively for the HIVE platform, as well as commonly used external tools adapted to operate within the parallel architecture of the system. HIVE is composed of a flexible infrastructure, which allows for simple implementation of new algorithms and tools. Currently, available HIVE tools include sequence alignment and nucleotide variation profiling tools, metagenomic analyzers, phylogenetic tree-building tools using NGS data, clone discovery algorithms, and recombination analysis algorithms. In addition to tools, HIVE also provides knowledgebases that can be used in conjunction with the tools for NGS sequence and metadata analysis.
Abstract: Overweight and obesity are major problems in today’s society, driving the prevalence of diabetes and its related complications. It is important to understand the molecular mechanisms underlying the chronic complications in diabetes in order to develop better therapeutic approaches for these conditions. Some of the most important complications include macrovascular abnormalities, e.g., heart disease and atherosclerosis, and microvascular abnormalities, e.g., retinopathy, nephropathy and neuropathy, in particular diabetic foot ulceration. The highly conserved endogenous small non-coding RNA molecules, the micro RNAs (miRNAs) have in recent years been found to be involved in a number of biological processes, including the pathogenesis of disease. Their main function is to regulate post-transcriptional gene expression by binding to their target messenger RNAs (mRNAs), leading to mRNA degradation, suppression of translation or even gene activation. These molecules are promising therapeutic targets and demonstrate great potential as diagnostic biomarkers for disease. This review aims to describe the most recent findings regarding the important roles of miRNAs in diabetes and its complications, with special attention given to the different phases of diabetic wound healing.