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1.
Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA.
Yang, EW, Bahn, JH, Hsiao, EY, Tan, BX, Sun, Y, Fu, T, Zhou, B, Van Nostrand, EL, Pratt, GA, Freese, P, et al
Nature communications. 2019;(1):1338
Abstract
Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.
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2.
RNA regulons are essential in intestinal homeostasis.
Parham, LR, Williams, PA, Chatterji, P, Whelan, KA, Hamilton, KE
American journal of physiology. Gastrointestinal and liver physiology. 2019;(1):G197-G204
Abstract
Intestinal epithelial cells are among the most rapidly proliferating cell types in the human body. There are several different subtypes of epithelial cells, each with unique functional roles in responding to the ever-changing environment. The epithelium's ability for rapid and customized responses to environmental changes requires multitiered levels of gene regulation. An emerging paradigm in gastrointestinal epithelial cells is the regulation of functionally related mRNA families, or regulons, via RNA-binding proteins (RBPs). RBPs represent a rapid and efficient mechanism to regulate gene expression and cell function. In this review, we will provide an overview of intestinal epithelial RBPs and how they contribute specifically to intestinal epithelial stem cell dynamics. In addition, we will highlight key gaps in knowledge in the global understanding of RBPs in gastrointestinal physiology as an opportunity for future studies.
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3.
Gene-function studies in systemic lupus erythematosus.
Rosetti, F, de la Cruz, A, Crispín, JC
Current opinion in rheumatology. 2019;(2):185-192
Abstract
PURPOSE OF REVIEW The aim of this review is to discuss recent developments in our understanding of how systemic lupus erythematosus (SLE)-associated genes contribute to autoimmunity. RECENT FINDINGS Gene-function studies have revealed mechanisms through which SLE-associated alleles of IFIH1, TNFAIP3, IRF5, and PRDM1 likely contribute to the development of autoimmunity. Novel research has identified Mac-1 (encoded by ITGAM), CaMK4, and iRhom2 as plausible therapeutic targets in lupus nephritis. SUMMARY The work discussed in this review has broad implications for our understanding of the pathogenesis of SLE and for the development of novel therapeutic strategies.
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4.
Classification of the nucleolytic ribozymes based upon catalytic mechanism.
Lilley, DMJ
F1000Research. 2019
Abstract
The nucleolytic ribozymes carry out site-specific RNA cleavage reactions by nucleophilic attack of the 2'-oxygen atom on the adjacent phosphorus with an acceleration of a million-fold or greater. A major part of this arises from concerted general acid-base catalysis. Recent identification of new ribozymes has expanded the group to a total of nine and this provides a new opportunity to identify sub-groupings according to the nature of the general base and acid. These include nucleobases, hydrated metal ions, and 2'-hydroxyl groups. Evolution has selected a number of different combinations of these elements that lead to efficient catalysis. These differences provide a new mechanistic basis for classifying these ribozymes.
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5.
Advances in CRISPR-Cas systems for RNA targeting, tracking and editing.
Wang, F, Wang, L, Zou, X, Duan, S, Li, Z, Deng, Z, Luo, J, Lee, SY, Chen, S
Biotechnology advances. 2019;(5):708-729
Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.
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6.
Role of amino acid residues important for nucleic acid binding in human Translin.
Gupta, A, Pillai, VS, Chittela, RK
The international journal of biochemistry & cell biology. 2019;:105593
Abstract
Translin is a multifunctional DNA/RNA binding protein involved in DNA repair and RNA metabolism. It has two basic regions and involvement of some residues in these regions in nucleic acid binding is established experimentally. Here we report the functional role of four residues of basic region II, Y85, R86, H88, R92 and one residue of C terminal region, K193 in nucleic acid binding using substitution mutant variants. CD analysis of the mutant proteins showed that secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Octameric state was maintained in all the mutants of basic region as evidenced by TEM, DLS, native PAGE and gel filtration analyses. However, K193G mutation completely abolished the octameric state of Translin protein and consequently its ability to bind ssDNA/ssRNA. The mutants of the basic region II exhibited a differential effect on nucleic acid binding, with R86A and R92G as most deleterious. Interestingly, H88A mutant showed higher nucleic acid binding affinity in comparison to the wild type Translin. An in silico analysis of the mutant variant sequences predicted all the mutations to be destabilizing, causing increase in flexibility and also leading to disruption of local interactions. The differential effect of mutations on DNA/RNA binding where octameric state is maintained could be attributed to these predicted disturbances.
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7.
High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries.
Lietard, J, Schaudy, E, Hölz, K, Ameur, D, Somoza, MM
Journal of visualized experiments : JoVE. 2019;(150)
Abstract
Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid·ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of β-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.
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8.
The functional consequences of sodium channel NaV 1.8 in human left ventricular hypertrophy.
Ahmad, S, Tirilomis, P, Pabel, S, Dybkova, N, Hartmann, N, Molina, CE, Tirilomis, T, Kutschka, I, Frey, N, Maier, LS, et al
ESC heart failure. 2019;(1):154-163
Abstract
AIMS: In hypertrophy and heart failure, the proarrhythmic persistent Na+ current (INaL ) is enhanced. We aimed to investigate the electrophysiological role of neuronal sodium channel NaV 1.8 in human hypertrophied myocardium. METHODS AND RESULTS Myocardial tissue of 24 patients suffering from symptomatic severe aortic stenosis and concomitant significant afterload-induced hypertrophy with preserved ejection fraction was used and compared with 12 healthy controls. We performed quantitative real-time PCR and western blot and detected a significant up-regulation of NaV 1.8 mRNA (2.34-fold) and protein expression (1.96-fold) in human hypertrophied myocardium compared with healthy hearts. Interestingly, NaV 1.5 protein expression was significantly reduced in parallel (0.60-fold). Using whole-cell patch-clamp technique, we found that the prominent INaL was significantly reduced after addition of novel NaV 1.8-specific blockers either A-803467 (30 nM) or PF-01247324 (1 μM) in human hypertrophic cardiomyocytes. This clearly demonstrates the relevant contribution of NaV 1.8 to this proarrhythmic current. We observed a significant action potential duration shortening and performed confocal microscopy, demonstrating a 50% decrease in proarrhythmic diastolic sarcoplasmic reticulum (SR)-Ca2+ leak and SR-Ca2+ spark frequency after exposure to both NaV 1.8 inhibitors. CONCLUSIONS We show for the first time that the neuronal sodium channel NaV 1.8 is up-regulated on mRNA and protein level in the human hypertrophied myocardium. Furthermore, inhibition of NaV 1.8 reduced augmented INaL , abbreviated the action potential duration, and decreased the SR-Ca2+ leak. The findings of our study suggest that NaV 1.8 could be a promising antiarrhythmic therapeutic target and merits further investigation.
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9.
Droplet-based single cell RNAseq tools: a practical guide.
Salomon, R, Kaczorowski, D, Valdes-Mora, F, Nordon, RE, Neild, A, Farbehi, N, Bartonicek, N, Gallego-Ortega, D
Lab on a chip. 2019;(10):1706-1727
Abstract
Droplet based scRNA-seq systems such as Drop-seq, inDrop and Chromium 10X have been the catalyst for the wide adoption of high-throughput scRNA-seq technologies in the research laboratory. In order to understand the capabilities of these systems to deeply interrogate biology; here we provide a practical guide through all the steps involved in a typical scRNA-seq experiment. Through comparing and contrasting these three main droplet based systems (and their derivatives), we provide an overview of all critical considerations in obtaining high quality and biologically relevant data. We also discuss the limitations of these systems and how they fit into the emerging field of Genomic Cytometry.
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10.
Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression.
Shen, X, Beasley, S, Putman, JN, Li, Y, Prakash, TP, Rigo, F, Napierala, M, Corey, DR
RNA (New York, N.Y.). 2019;(9):1118-1129
Abstract
Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells.