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Metabolic Incorporation of Azide Functionality into Cellular RNA.

ABSTRACT: Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

SUBMITTER: Nainar S

PROVIDER: S-EPMC5115926 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Metabolic Incorporation of Azide Functionality into Cellular RNA.

Nainar Sarah S Beasley Samantha S Fazio Michael M Kubota Miles M Dai Nan N Corrêa Ivan R IR Spitale Robert C RC

Chembiochem : a European journal of chemical biology 20160930 22

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further dem ...[more]

PMID: 27595557

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Project description:BackgroundExpansins are relatively small proteins that lack enzymatic activity and are found in plants and microorganisms. The function of these proteins is to disrupt the plant cell walls by interfering with the non-covalent interchain bonding of the polysaccharides. Expansins were found to be important for plant growth, but they are also expressed by various bacteria known to have interactions with plants. Clostridium clariflavum is a plant cell wall-degrading bacterium with a highly elaborate cellulosomal system. Among its numerous dockerin-containing genes, two expansin-like proteins, Clocl_1862 and Clocl_1298 (termed herein CclEXL1 and CclEXL2) were identified, and CclEXL1 was found to be expressed as part of the cellulosome system. This is the first time that an expansin-like protein is identified in a cellulosome complex, which implicates its possible role in biomass deconstruction.ResultsIn the present article, we analyzed the functionality of CclEXL1. Its dockerin was characterized and shown to bind selectively to type-I cohesins of C. clariflavum, with preferential binding to the cohesin of ScaG, and additionally to a type-I cohesin of C. cellulolyticum. We demonstrated experimentally that the expansin-like protein binds preferentially to microcrystalline cellulose, but it also binds to acid-swollen cellulose, xylan, and wheat straw. CclEXL1 exhibited a pronounced loosening effect on filter paper, which resulted in substantial decrease in tensile stress. The C. clariflavum expansin-like protein thus enhances significantly enzymatic hydrolysis of cellulose, both by C. clariflavum cellulosomes and two major cellulosomal cellulases from this bacterium: GH48 (exoglucanase) and GH9 (endoglucanase). Finally, we demonstrated CclEXL1-mediated enhancement of microcrystalline cellulose degradation by different cellulosome fractions and the two enzymes.ConclusionsThe results of this study confirm that the C. clariflavum expansin-like protein is part of the elaborate cellulosome system of this bacterium with capabilities of cellulose creeping. The data suggest that pretreatment of cellulosic materials with CclEXL1 can bring about substantial improvement of hydrolysis by cellulases.

Project description:Regulatory T cells (Treg) are well-known for their immune regulatory potential and are essential for maintaining immune homeostasis. The rationale of Treg-based immunotherapy for treating autoimmunity and transplant rejection is to tip the immune balance of effector T cell-mediated immune activation and Treg-mediated immune inhibition in favor of Treg cells, either through endogenous Treg expansion strategies or adoptive transfer of ex vivo expanded Treg. Compelling evidence indicates that Treg show properties of phenotypic heterogeneity and instability, which has caused considerable debate in the field regarding their correct use. Consequently, for further optimization of Treg-based immunotherapy, it is vital to further our understanding of Treg proliferative, migratory, and suppressive behavior. It is increasingly appreciated that the functional profile of immune cells is highly dependent on their metabolic state. Although the metabolic profiles of effector T cells are progressively understood, little is known on Treg in this respect. The objective of this review is to outline the current knowledge of human Treg metabolic profiles associated with the regulation of Treg functionality. As such information on human Treg is still limited, where information was lacking, we included insightful findings from mouse studies. To assess the available evidence on metabolic pathways involved in Treg functionality, PubMed, and Embase were searched for articles in English indexed before April 28th, 2019 using "regulatory T lymphocyte," "cell metabolism," "cell proliferation," "migration," "suppressor function," and related search terms. Removal of duplicates and search of the references was performed manually. We discerned that while glycolysis fuels the biosynthetic and bioenergetic needs necessary for proliferation and migration of human Treg, suppressive capacity is mainly maintained by oxidative metabolism. Based on the knowledge of metabolic differences between Treg and non-Treg cells, we additionally discuss and propose ways of how human Treg metabolism could be exploited for the betterment of tolerance-inducing therapies.

Dataset Information

Metabolic Incorporation of Azide Functionality into Cellular RNA.

Publications

Metabolic Incorporation of Azide Functionality into Cellular RNA.

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