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The mutual interactions of RNA, counterions and water - quantifying the electrostatics at the phosphate-water interface.
Fingerhut, BP
Chemical communications (Cambridge, England). 2021;(96):12880-12897
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Abstract
The structure and dynamics of polyanionic biomolecules, like RNA, are decisively determined by their electric interactions with the water molecules and the counterions in the environment. The solvation dynamics of the biomolecules involves a subtle balance of non-covalent and many-body interactions with structural fluctuations due to thermal motion occurring in a femto- to subnanosecond time range. This complex fluctuating many particle scenario is crucial in defining the properties of biological interfaces with far reaching significance for the folding of RNA structures and for facilitating RNA-protein interactions. Given the inherent complexity, suited model systems, carefully calibrated and benchmarked by experiments, are required to quantify the relevant interactions of RNA with the aqueous environment. In this feature article we summarize our recent progress in the understanding of the electrostatics at the biological interface of double stranded RNA (dsRNA) and transfer RNA (tRNA). Dimethyl phosphate (DMP) is introduced as a viable and rigorously accessible model system allowing the interaction strength with water molecules and counterions, their relevant fluctuation timescales and the spatial reach of interactions to be established. We find strong (up to ≈90 MV cm-1) interfacial electric fields with fluctuations extending up to ≈20 THz and demonstrate how the asymmetric stretching vibration νAS(PO2)- of the polarizable phosphate group can serve as the most sensitive probe for interfacial interactions, establishing a rigorous link between simulations and experiment. The approach allows for the direct interfacial observation of interactions of biologically relevant Mg2+ counterions with phosphate groups in contact pair geometries via the rise of a new absorption band imposed by exchange repulsion interactions at short interatomic distances. The systematic extension to RNA provides microscopic insights into the changes of the hydration structure that accompany the temperature induced melting of the dsRNA double helix and quantify the ionic interactions in the folded tRNA. The results show that pairs of negatively charged phosphate groups and Mg2+ ions represent a key structural feature of RNA embedded in water. They highlight the importance of binding motifs made of contact pairs in the electrostatic stabilization of RNA structures that have a strong impact on the surface potential and enable the fine tuning of the local electrostatic properties which are expected to be relevant for mediating the interactions between biomolecules.
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DExD/H-box helicases: multifunctional regulators in antiviral innate immunity.
Su, C, Tang, YD, Zheng, C
Cellular and molecular life sciences : CMLS. 2021;(1):2
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Abstract
DExD/H-box helicases play critical roles in multiple cellular processes, including transcription, cellular RNA metabolism, translation, and infections. Several seminal studies over the past decades have delineated the distinct functions of DExD/H-box helicases in regulating antiviral innate immune signaling pathways, including Toll-like receptors, retinoic acid-inducible gene I-like receptors, cyclic GMP-AMP synthase-the stimulator of interferon gene, and NOD-like receptors signaling pathways. Besides the prominent regulatory roles, there is increasing attention on their functions as nucleic acid sensors involved in antiviral innate immunity. Here we summarize the complex regulatory roles of DExD/H-box helicases in antiviral innate immunity. A better understanding of the underlying molecular mechanisms of DExD/H-box helicases' regulatory roles is vital for developing new therapeutics targeting DExD/H-box helicases and their mediated signaling transduction in viral infectious diseases.
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RNA Proximity Labeling: A New Detection Tool for RNA-Protein Interactions.
Weissinger, R, Heinold, L, Akram, S, Jansen, RP, Hermesh, O
Molecules (Basel, Switzerland). 2021;(8)
Abstract
Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA-protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA-protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.
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The RNA m6A writer METTL14 in cancers: Roles, structures, and applications.
Zhou, H, Yin, K, Zhang, Y, Tian, J, Wang, S
Biochimica et biophysica acta. Reviews on cancer. 2021;(2):188609
Abstract
N6-methyladenosine (m6A) is the most abundant and diverse epigenetic modification of mRNAs in eukaryotes, and it regulates biological metabolism, cell differentiation and cycles, and responses to heat shock stress, cancers and other diseases. RNA methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14) and other proteins possessing methyltransferase (MTase) capability including Wilms tumor 1-associated protein (WTAP), RNA-binding motif protein 15(RBM15), KIAA1429 and zinc finger CCCH-type containing 13 (ZC3H13) constitute the m6A writer complex. Although METTL3 is the catalytic subunit, its activity is strongly dependent on METTL14, which is crucial in maintaining complex integrity and recognizing special RNA substrates. Currently, the roles of m6A modification in cancers are being extensively reviewed. The critical functions of METTL14 in the occurrence and development of a variety of cancers as well as the potential targeting of METTL14 as a cancer treatment have not yet been highlighted. Therefore, in this review, we summarize the m6A modification and focus on the structure and functions of METTL14 as well as its roles in oncogenesis, metastasis progression, treatment and prognosis in cancer.
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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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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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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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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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9.
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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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.