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1.
Reversal of nucleobase methylation by dioxygenases.
Xu, GL, Bochtler, M
Nature chemical biology. 2020;(11):1160-1169
Abstract
The repertoire of nucleobase methylation in DNA and RNA, introduced by chemical agents or enzymes, is large. Most methylation can be reversed either directly by restoration of the original nucleobase or indirectly by replacement of the methylated nucleobase with an unmodified nucleobase. In many direct and indirect demethylation reactions, ALKBH (AlkB homolog) and TET (ten eleven translocation) hydroxylases play a role. Here, we suggest a chemical classification of methylation types. We then discuss pathways for removal, emphasizing oxidation reactions. We highlight the recently expanded repertoire of ALKBH- and TET-catalyzed reactions and describe the discovery of a TET-like protein that resembles the hydroxylases but uses an alternative co-factor and catalyzes glyceryl transfer rather than hydroxylation.
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2.
Postsynthetic Modifications of DNA and RNA by Means of Copper-Free Cycloadditions as Bioorthogonal Reactions.
Krell, K, Harijan, D, Ganz, D, Doll, L, Wagenknecht, HA
Bioconjugate chemistry. 2020;(4):990-1011
Abstract
Bioorthogonal chemistry has mainly been developed for proteins and carbohydrates. The chemistry of nucleic acids is different, and bioorthogonal labeling strategies that were successfully applied for proteins and carbohydrates cannot be simply transferred to DNA and RNA. Cycloadditions play a central role for bioorthogonal chemistry with nucleic acids. In vivo postsynthetic labeling of DNA and RNA requires copper-free variants of cycloaddition chemistry to achieve "bio"orthogonality that can be applied even in living cells. Currently, there are three major types of copper-free cycloadditions available for nucleic acids: (i) the ring-strain-promoted azide-alkyne cycloadditions, (ii) the "photoclick" 1,3-dipolar cycloadditions, and (iii) the Diels-Alder reactions with inverse electron demand. In principle, bioorthogonally reactive building blocks for postsynthetic modifications of nucleic acids by cycloaddition can be prepared by three different ways: (i) The organic synthesis of DNA and RNA applies phosphoramidites as building blocks for solid-phase automated chemistry. (ii) The biochemical preparation of DNA and RNA by primer extension (PEX) and PCR applies triphosphates as building blocks together with DNA/RNA polymerases, and works in aqueous buffer. (iii) DNA and RNA is labeled by the intrinsic metabolism in cells using bioorthogonally reactive nucleosides. In contrast to proteins and carbohydrates, for which metabolic labeling strategies are well developed, there are only a few examples in the literature for metabolic labeling of nucleic acids. In this review, we summarize the currently available DNA and RNA building blocks, both phosphoramidites and nucleotide triphosphates, for copper-free and bioorthogonal postsynthetic modification strategies.
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3.
Genetic alphabet expansion technology by creating unnatural base pairs.
Kimoto, M, Hirao, I
Chemical Society reviews. 2020;(21):7602-7626
Abstract
Recent advancements in the creation of artificial extra base pairs (unnatural base pairs, UBPs) are opening the door to a new research area, xenobiology, and genetic alphabet expansion technologies. UBPs that function as third base pairs in replication, transcription, and/or translation enable the site-specific incorporation of novel components into DNA, RNA, and proteins. Here, we describe the UBPs developed by three research teams and their application in PCR-based diagnostics, high-affinity DNA aptamer generation, site-specific labeling of RNAs, semi-synthetic organism creation, and unnatural-amino-acid-containing protein synthesis.
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4.
The Autophagy-RNA Interplay: Degradation and Beyond.
Abildgaard, MH, Brynjólfsdóttir, SH, Frankel, LB
Trends in biochemical sciences. 2020;(10):845-857
Abstract
Autophagy is a highly conserved degradation pathway that ensures nutrient recycling and removal of unwanted substrates. This process has a fundamental role in stress adaptation and maintenance of cellular homeostasis. Here, we discuss emerging aspects of the autophagy-RNA interplay, including autophagy-mediated degradation of RNA, RNA-binding proteins (RBPs), and ribonucleoprotein (RNP) complexes. Beyond degradation, we review new roles for autophagy players in the secretion and intracellular transport of RNA and related complexes. We discuss the physiological importance of these events for RNA homeostasis and gene expression programs, as well as their implications for disease, including cancer and neurodegeneration. Lastly, we examine how post-transcriptional regulation of autophagy, through specialized processing and selective translation of key transcripts, challenges and updates our current view of autophagy complexity.
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5.
Expression of Fibulin-2 and Fibulin-5 on subretinal fluid in human primary rhegmatogenous retinal detachment.
Davila-Avila, N, Muñiz-Ruvalcaba, FP, Hernandez-Zimbron, LF, Gonzalez-Salinas, R, Corredor-Ortega, C, Perez-Vazquez, J, Soberon, S, Quiroz-Mercado, H
Experimental eye research. 2020;:107992
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6.
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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7.
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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8.
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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9.
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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10.
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.