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In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

PROTEIN SCIENCE 2017年第7期26卷 1363-1379页

作者： Custer, Thomas C. Walter, Nils G. Univ Michigan Program Chem Biol Ann Arbor MI 48109 USA Univ Michigan Dept Chem Single Mol Anal Grp Ann Arbor MI 48109 USA Univ Michigan Dept Chem Ctr RNA Biomed Ann Arbor MI 48109 USA

rna plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexesmolecular machinesused to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of rnas involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular rna functions, including those of protein coding messenger rnas (mrnas). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label rna molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 rna polymerase to body label an rna with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mrna, whereas protein coding is severely diminished in the case of body labeling.

关键词： single-molecule fluorescence imaging noncoding rna fluorophore labeling single particle tracking

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Phytochemicals modulate carcinogenic signaling pathways in breast and hormone-related cancers

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ONCOTARGETS AND THERAPY 2015年 8卷 2053-2066页

作者： Petric, Roxana Cojocneanu Braicu, Cornelia Raduly, Lajos Zanoaga, Oana Dragos, Nicolae Monroig, Paloma Dumitrascu, Dan Berindan-Neagoe, Ioana Univ Babes Bolyai Dept Biol R-3400 Cluj Napoca Romania Iuliu Hatieganu Univ Med & Pharm Res Ctr Funct Genom Biomed & Translat Med Cluj Napoca 400337 Romania Univ Agr Sci & Vet Med Fac Vet Med Dept Physiopathol Cluj Napoca Romania Inst Biol Res Dept Taxon & Ecol Cluj Napoca Romania Univ Texas MD Anderson Canc Ctr Dept Expt Therapeut Houston TX 77030 USA Iuliu Hatieganu Univ Med & Pharm Dept Internal Med 2 Cluj Napoca 400337 Romania Oncol Inst Prof Dr Ion Chiricuta Dept Funct Genom & Expt Pathol Cluj Napoca Romania Iuliu Hatieganu Univ Med & Pharm Dept Immunol Cluj Napoca 400337 Romania

Over the years, nutrition and environmental factors have been demonstrated to influence human health, specifically cancer. Owing to the fact that cancer is a leading cause of death worldwide, efforts are being made to elucidate molecular mechanisms that trigger or delay carcinogenesis. Phytochemicals, in particular, have been shown to modulate oncogenic processes through their antioxidant and anti-inflammatory activities and their ability to mimic the chemical structure and activity of hormones. These compounds can act not only by influencing oncogenic proteins, but also by modulating noncoding rnas such as micrornas and long noncoding rnas. Although we are only beginning to understand the complete effects of many natural compounds, such as phytochemicals, researchers are motivated to combine these agents with traditional, chemo-based, or hormone-based therapies to fight against cancer. Since ongoing studies continue to prove effective, herein we exalt the importance of improving dietary choices as a chemo-preventive strategy.

关键词： natural compounds dietary polyphenols epigallocatechin gallate curcumin genistein noncoding rna cancer

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Yrna Expression Profiles are Altered in Clear Cell Renal Cell Carcinoma

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EUROPEAN UROLOGY FOCUS 2018年第2期4卷 260-266页

作者： Nientiedt, Malin Schmidt, Doris Kristiansen, Glen Mueller, Stefan C. Ellinger, Joerg Univ Hosp Bonn Dept Urol Bonn Germany Univ Hosp Bonn Inst Pathol Bonn Germany

Background: noncoding rnas play an important role in human carcinogenesis. Yrnas, a novel class of noncoding rnas, have been identified as biomarkers in breast cancer patients. Objective: To test the hypothesis that Yrna expression is dysregulated in clear cell renal cell carcinoma (ccRCC). Design, setting, and participants: We first measured the expression of all known Yrnas (hY1, hY3, hY4, and hY5) in a screening cohort (30 ccRCC and 15 normal renal tissues). Subsequently, hY3 and hY4 were validated in an independent cohort (88 ccRCC and 59 normal renal tissues). Finally, hY3 and hY4 levels in serum samples from 30 ccRCC and 15 control individuals were measured. Yrnas were detected using quantitative real-time polymerase chain reaction. Outcome measurements and statistical analysis: Relative expression values were analyzed using the Mann-Whitney-U test and Kaplan Meier estimates. Results and limitations: Expression of hY3 and hY4 was increased in ccRCC samples compared with normal renal tissue, whereas hY1 and hY5 levels were similar. Expression levels of hY4 correlated with ccRCC stage and the presence of lymph node metastases. Neither hY3 nor hY4 were circulating at different levels in ccRCC patients and control individuals. Conclusions: The expression of hY3 and hY4 is altered in ccRCC and associated with advanced disease. Patient summary: In this report we studied the expression of noncoding Yrna in clear cell renal cell carcinoma tissue. We observed increased hY3 and hY4 expression levels in cancer tissues. However, expression levels were not different in the serum of patients with cancer or benign disease. (C) 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

关键词： Renal cell carcinoma Yrna noncoding rna Gene expression

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Statistical analysis of non-coding rna data

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CANCER LETTERS 2018年 417卷 161-167页

作者： He, Qianchuan Liu, Yang Sun, Wei Fred Hutchinson Canc Res Ctr Div Publ Hlth Sci Seattle WA 98109 USA

With rapid progress in high-throughput genome technology, the study of noncoding rna has arisen as a highly popular topic in biomedical research. noncoding rna plays fundamental roles in cell proliferation, cell differentiation and epigenetic regulation, and the study of noncoding rna will yield novel insights into gene regulation and provide new clues for disease treatment. However, due to the large volume and diverse functions of noncoding rnas, the analysis of these rnas has proved to be a challenging task. In this review, we review the commonly used computational tools for the identification of noncoding rnas, and discuss popular statistical tools for their analysis. Due to the large body of noncoding rna classes, we focus on the analysis of microrna and long noncoding rna, two of the most widely studied classes of noncoding rnas. Specific examples are provided to show the context of the analysis. This review aims to provide up-to-date information on existing tools and methods for identifying and analyzing noncoding rna. (C) 2018 Elsevier B.V. All rights reserved.

关键词： Long noncoding rna Microrna noncoding rna Statistical analysis Statistical modeling Target prediction

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A Structural Basis for the Recognition of 2′-Deoxyguanosine by the Purine Riboswitch

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JOUrnaL OF MOLECULAR BIOLOGY 2009年第3期385卷 938-948页

作者： Edwards, Andrea L. Batey, Robert T. Univ Colorado Dept Chem & Biochem Boulder CO 80309 USA

Riboswitches are noncoding rna elements that are commonly found in the 5'-untranslated region of bacterial mrna. Binding of a small-molecule metabolite to the riboswitch aptamer domain guides the folding of the downstream sequence into one of two mutually exclusive secondary structures that directs gene expression. The purine riboswitch family, which regulates aspects of purine biosynthesis and transport, contains three distinct classes that specifically recognize guanine/hypoxanthine, adenine, or 2'-deoxyguanosine (dG). Structural analysis of the guanine and adenine classes revealed a binding pocket that almost completely buries the nucleobase within the core of the folded rna. Thus, it is somewhat surprising that this family of rna elements also recognizes dG. We have used a combination of structural and biochemical techniques to understand how the guanine riboswitch could be converted into a dG binder and the structural basis for dG recognition. These studies reveal that a limited number of sequence changes to a guanine-sensing rna are required to cause a specificity switch from guanine to 2'-deoxyguanosine, and to impart an altered structure for accommodating the additional deoxyribose sugar moiety. (C) 2008 Elsevier Ltd. All rights reserved.

关键词： rna-ligand interaction riboswitch X-ray crystallography isothermal titration calorimetry noncoding rna

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Epigenetic factors as drivers of fibrosis in systemic sclerosis

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EPIGENOMICS 2017年第4期9卷 463-477页

作者： Bergmann, Christina Distler, Joerg H. W. Friedrich Alexander Univ Nurnberg FAU Dept Internal Med 3 Erlangen Germany Univ Hosp Erlangen Erlangen Germany

Prolonged activation of fibroblasts is a central hallmark of fibrosing disorders such as systemic sclerosis (SSc). Fibroblasts are the key effector cells. They differentiate into an activated myofibroblast phenotype. In contrast to normal wound healing with transient activation, myofibroblasts persist in fibrosing disorders. Current hypothesis suggests that profibrotic cytokines might trigger epigenetic changes which contribute to the persistently activated fibroblast phenotype. In the last years, several epigenetic alterations have been described in SSc and have been linked to different pathogenic aspects of the disease, in particular to aberrant fibroblast activation and tissue fibrosis, but also to vascular manifestations and inflammation. The focus of this review is the current knowledge on epigenetic changes in fibroblast activation in SSc.

关键词： DNA-methylation epigenetics histone modification myofibroblast differentiation noncoding rna systemic sclerosis

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Ageing-associated small rna cargo of extracellular vesicles

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rna BIOLOGY 2023年第1期20卷 482-494页

作者： Kern, Fabian Kuhn, Thomas Ludwig, Nicole Simon, Martin Groeger, Laura Fabis, Natalie Aparicio-Puerta, Ernesto Salhab, Abdulrahman Fehlmann, Tobias Hahn, Oliver Engel, Annika Wagner, Viktoria Koch, Marcus Winek, Katarzyna Soreq, Hermona Nazarenko, Irina Fuhrmann, Gregor Wyss-Coray, Tony Meese, Eckart Keller, Verena Laschke, Matthias W. Keller, Andreas Saarland Univ Chair Clin Bioinformat Saarland Informat Campus D-66123 Saarbrucken Germany Helmholtz Ctr Infect Res HZI Helmholtz Inst Pharmaceut Res Saarland HIPS Dept Clin Bioinformat Saarbrucken Germany Helmholtz Ctr Infect Res HZI Helmholtz Inst Pharmaceut Res Saarland HIPS Biogen Nanotherapeut Grp ION Saarbrucken Germany Saarland Univ Dept Pharm Saarbrucken Germany Saarland Univ Dept Human Genet Homburg Germany Saarland Univ Ctr Human & Mol Biol Homburg Germany Wuppertal Univ Mol Cell Biol & Microbiol Wuppertal Germany Saarland Univ Dept Genet & Epigenet Saarbrucken Germany Stanford Univ Dept Neurol & Neurol Sci Stanford CA USA INM Leibniz Inst New Mat Saarbrucken Germany Hebrew Univ Jerusalem Edmond & Lily Safra Ctr Brain Sci Jerusalem Israel Univ Freiburg Inst Infect Prevent & Control Fac Med Freiburg Germany Univ Freiburg Med Ctr Freiburg Germany Saarland Univ Inst Clin & Expt Surg Homburg Germany Saarland Univ Ctr Bioinformat Saarbrucken Germany

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of rnas. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory rnas (sncrnas) in two mouse plasma fractions at five time points across the lifespan from 2-18 months: (1) sncrnas that are free-circulating (fc-rna) and (2) sncrnas bound outside or inside EVs (EV-rna). Different sncrna classes exhibit unique ageing patterns that vary between the fcrna and EV-rna fractions. While trnas showed the highest correlation with ageing in both fractions, rrnas exhibited inverse correlation trajectories between the EV- and fc-fractions. For mirnas, the EV-rna fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncrnas and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.

关键词： noncoding rna tissue ageing extracellular vesicles plasma mirna >

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Grad-seq guides the discovery of ProQ as a major small rna-binding protein

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2016年第41期113卷 11591-11596页

作者： Smirnov, Alexandre Foerstner, Konrad U. Holmqvist, Erik Otto, Andreas Guenster, Regina Becher, Doerte Reinhardt, Richard Vogel, Joerg Univ Wurzburg Inst Mol Infect Biol RNA Biol Grp D-97080 Wurzburg Germany Univ Wrzburg Core Unit Syst Med D-97080 Wurzburg Germany Ernst Moritz Arndt Univ Greifswald Inst Microbiol D-17489 Greifswald Germany Max Planck Genome Ctr Cologne Max Planck Inst Plant Breeding Res D-50829 Cologne Germany Univ Wurzburg Res Ctr Infect Dis ZINF D-97070 Wurzburg Germany

The functional annotation of transcriptomes and identification of noncoding rna (ncrna) classes has been greatly facilitated by the advent of next-generation rna sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncrnas dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative rna landscape of organisms with potentially new rna biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of rnas with functionally important cognate rna-binding proteins. Here we analyze the full ensemble of cellular rnas using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of rna-protein interactions. In addition to capturing established rna classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small rnas that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant rna-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional rna landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional rna classes and major rna-binding proteins in both model species and genetically intractable organisms.

关键词： small rna noncoding rna rna-protein interaction ProQ Hfq

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Epigenetic signatures in cardiac fibrosis, special emphasis on DNA methylation and histone modification

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HEART FAILURE REVIEWS 2018年第5期23卷 789-799页

作者： Tao, Hui Song, Zheng-Yu Ding, Xuan-Sheng Yang, Jing-Jing Shi, Kai-Hu Li, Jun China Pharmaceut Univ Sch Basic Med Sci & Clin Pharm Nanjing 210009 Jiangsu Peoples R China Nanjing Univ Chinese Med Affiliated Hosp Integrated Tradit Chinese & Weste Dept Cardiothorac Surg Nanjing 210028 Jiangsu Peoples R China Anhui Med Univ Hosp 2 Dept Pharmacol Hefei 230601 Anhui Peoples R China Anhui Med Univ Sch Pharm Hefei 230032 Anhui Peoples R China

Cardiac fibrosis is defined as excess deposition of extracellular matrix (ECM), resulting in tissue scarring and organ dysfunction. In recent years, despite the underlying mechanisms of cardiac fibrosis are still unknown, numerous studies suggest that epigenetic regulation of cardiac fibrosis. Cardiac fibrosis is regulated by a myriad of factors that converge on the transcription of genes encoding extracellular matrix protein, a process the epigenetic machinery plays a pivotal role. Epigenetic modifications contain three main processes: DNA methylation, histone modifications, and noncoding rnas. Here, we review recent studies that have illustrated key roles for epigenetic events in the control of pro-fibrotic gene expression, and highlight the potential of molecule mechanisms that target epigenetic regulators as a means of treating cardiac fibrosis.

关键词： Cardiac fibrosis Epigenetic DNA methylation noncoding rna Histone modification

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Small regulatory bacterial rnas regulating the envelope stress response

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BIOCHEMICAL SOCIETY TRANSACTIONS 2017年第2期45卷 417-425页

作者： Klein, Gracjana Raina, Satish Gdansk Univ Technol Unit Bacterial Genet Narutowicza 11-12 PL-80233 Gdansk Poland

Most bacteria encode a large repertoire of rna-based regulatory mechanisms. Recent discoveries have revealed that the expression of many genes is controlled by a plethora of base-pairing noncoding small regulatory rnas (srnas), regulatory rna-binding proteins and rna-degrading enzymes. Some of these rna-based regulated processes respond to stress conditions and are involved in the maintenance of cellular homeostasis. They achieve it by either direct posttranscriptional repression of several mrnas, including blocking access to ribosome and/or directing them to rna degradation when the synthesis of their cognate proteins is unwanted, or by enhanced translation of some key stress-regulated transcriptional factors. noncoding rnas that regulate the gene expression by binding to regulatory proteins/transcriptional factors often act negatively by sequestration, preventing target recognition. Expression of many srnas is positively regulated by stress-responsive sigma factors like RpoE and RpoS, and two-component systems like PhoP/Q, Cpx and Rcs. Some of these regulatory rnas act via a feedback mechanism on their own regulators, which is best reflected by recent discoveries, concerning the regulation of cell membrane composition by srnas in Escherichia coli and Salmonella, which are highlighted here.

关键词： RfaH RpoE sigma factor envelope stress noncoding rna stress response two-component system

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