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作者： Pedro Gabriel Nachtigall Luiz Augusto Bovolenta

Micrornas (mirnas) are small noncoding rnas that are recognized as posttranscriptional regulators of gene expression. These molecules have been shown to play important roles in several cellular processes. Mirnas act on their target by guiding the RISC complex and binding to the mrna molecule. Thus, it is recognized that the function of a mirna is determined by the function of its target (s). By using high-throughput methodologies, novel mirnas are being identified, but their functions remain uncharted. Target validation is crucial to properly understand the specific role of a mirna in a cellular pathway. However, molecular techniques for experimental validation of mirna–target interaction are expensive, time-consuming, laborious, and can be not accurate in inferring true interactions. Thus, accurate mirna target predictions are helpful to understand the functions of mirnas. There are several algorithms proposed for target prediction and databases containing mirna-target information. However, these available computational tools for prediction still generate a large number of false positives and fail to detect a considerable number of true targets, which indicates the necessity of highly confident approaches to identify bona fide mirna–target interactions. This chapter focuses on tools and strategies used for mirna target prediction, by providing practical insights and outlooks. less

关键词： Bioinformatics Computational biology noncoding rna mirna recognition element Target prediction tools

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CRISPR-Cas9 Editing of the

CRISPR-Cas9 Editing of the

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作者： Xu Zhang Kirk William Deitsch Ron Dzikowski

The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system. less

关键词： CRISPR-Cas9 Genome Editing Parasite Culture Transfection Electroporation Microscopy Genome Editing Real-time PCR PCR Whole-genome Sequencing Genomics DNA Extraction Plasmodium falciparum Escherichia coli Chromatin Homologous recombination noncoding rna Telomere healing Mutually exclusive expression Multicopy gene families Subtelomeric domains

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Polyadenylation of Sine Transcripts Generated by rna Polymerase III Dramatically Prolongs Their Lifetime in Cells

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MOLECULAR BIOLOGY 2020年第1期54卷 67-74页

作者： Ustyantsev, I. G. Tatosyan, K. A. Stasenko, D., V Kochanova, N. Y. Borodulina, O. R. Kramerov, D. A. Russian Acad Sci Engelhardt Inst Mol Biol Moscow 119991 Russia

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various trnas and less often from 5S rrna and 7SL rna. Similar to the genes of these rnas, SINEs are transcribed by rna polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the rna polymerase III transcripts. It is well known that this polyadenylation of many rna polymerase II transcripts (e.g., mrnas) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20-30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the rna from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding rnas, which are stable in a cell due to polyadenylation.

关键词： SINE retroposones noncoding rna rna polymerase III polyadenylation poly(A) rna stability rna decay mammals

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Synergistic effects of carbon nanoparticle-Cr-Pb in PM2.5 cause cell cycle arrest via upregulating a novel lncrna NONHSAT074301.2 in human bronchial epithelial cells

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JOUrnaL OF HAZARDOUS MATERIALS 2021年 411卷 125070-125070页

作者： Pan, Xiujiao Wu, Jialong Jiang, Cuijuan Yu, Qianhui Yan, Bing Guangzhou Univ Key Lab Water Qual & Conservat Pearl River Delta Minist Educ Inst Environm Res Greater Bay Area Guangzhou 510006 Peoples R China Shandong Univ Sch Environm Sci & Engn Jinan 250100 Peoples R China

Inhalation of carcinogenic PM2.5 particles is a severe threat to all the people in both developing and developed nations. However, which components of PM2.5 and how they perturb human cells to cause various diseases are still not understood. Here, employing a reductionism approach, we revealed that one of the crucial toxic and pathogenic mechanisms of PM2.5 was the blocking of human bronchial cell cycle through upregulation of a novel long non-coding rna NONHSAT074301.2 by carbon particles with payloads of Cr(VI) and Pb2+. We also discovered that NONHSAT074301.2 is a key regulatory molecule controlling cell cycle arrest at G2/M phase. This work highlights cellular function and molecular signaling events investigations using a 16-membered combinational model PM2.5 library which contain carbon particles carrying four toxic pollutants in all possible combinations at environmental relevant concentrations. This work demonstrates a very powerful methodology to elucidate mechanisms at molecular level and help unlock the ?black box? of PM2.5-induced toxicities.

关键词： Combinatorial library Causal effect noncoding rna Synergistic effects Mechanistic toxicity

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Regulatory Roles of noncoding rna in Development and Disease

Regulatory Roles of Noncoding RNA in Development and Disease

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作者： GAURAV KUMAR PANDEY Uppsala University

学位级别：博士

Long noncoding rnas (lncrnas) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncrnas in development and disease model systems. Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncrna. Deletion of an 890bp region at the 5' end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 rna establishes CpG methylation by interacting with DNMT1. To explore the stability of lncrna mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an rna independent manner. To identify chromatin-associated lncrna (CARs) on a genome-wide scale, we purified rna from the sucrose gradient fractionated chromatin and subjected it to rna sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure. We have explored the functional role of lncrna in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncrnas differentially expressed between these subtypes. We report an uncharacterized rna NBAT-1, expressed at lower levels in high-risk tumors relative to low- risk tumors. Using neuroblastoma cell culture system, we

关键词： noncoding rna Genomic Imprinting Epigenetics Neuroblastoma

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Interrelation of Telomeres with Transposable Elements in Aging

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ADVANCES IN GERONTOLOGY 2020年第2期10卷 101-108页

作者： Mustafin, R. N. Bashkir State Med Univ Ufa 450008 Russia

The nature of telomere functioning, which affects the aging of the body, reflects global processes in the genome conditioned by the regulatory influence of transposable elements that are sequentially activated during ontogenesis. This is determined by the fact that centromeres and centromere proteins, telomeres and telomerases, introns and spliceosome components, transcription factors and their binding sites, noncoding rnas and their targets in protein-coding gene sequences evolutionarily originated from transposable elements. The interrelation of these structural and functional elements of the genome is dynamically changing in individual development and depends on the nature of activations and transpositions of transposable elements. Each species is characterized by a specific set of transposable elements and associated tandem repeats, which reflect the epigenetic tuning of ontogenesis, mainly as a part of centromeres and telomeres;the impact on them is promising for the development of mechanisms of lifespan regulation. This is due to the ability of ribozymes and peptides to interact with specific sequences of DNA nucleotides, especially in tandem repeats. An important approach to the study of the relationship of transposons with telomeres, centromeres, and subtelomeric regions for the regulation of aging may be the study of the role of the peptides and micrornas that unite them, the complex application of which has high potential for geroprotective effectiveness.

关键词： noncoding rna reverse transcriptase tandem repeats self-regulation telomeres

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An update on epigenetic regulation in autoimmune diseases

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JOUrnaL OF TRANSLATIONAL AUTOIMMUNITY 2022年 5卷

作者： Mei, Xiaole Zhang, Bo Zhao, Ming Lu, Qianjin Chinese Acad Med Sci & Peking Union Med Coll Inst Dermatol Nanjing Jiangsu Peoples R China Chinese Acad Med Sci Key Lab Basic & Translat Res Immunol Dermatol Nanjing Jiangsu Peoples R China Chinese Acad Med Sci & Peking Union Med Coll Inst Dermatol Jiangsu Key Lab Mol Biol Skin Dis & STTs Nanjing Jiangsu Peoples R China Chinese Acad Med Sci Res Unit Key Technol Diag & Treatment Immune Rela Changsha Hunan Peoples R China Cent South Univ Xiangya Hosp 2 Dept Dermatol Hunan Key Lab Med Epigen Changsha Hunan Peoples R China

Autoimmune diseases (AIDs) generally manifest as chronic immune disorders characterized by significant het-erogeneity and complex symptoms. The discordant incidence of AIDs between monozygotic twins guided people to attach importance to environmental factors. Epigenetics is one of the major ways to be influenced, some of them can even occur years before clinical diagnosis. With the advent of high-throughput omics times, the mysterious veil of epigenetic modification in AIDs has been gradually unraveled, and some progress has been made in utilizing it as indicators of diagnosis and disease activity. For example, the hypomethylated IFI44L promoter in diagnosing systematic lupus erythematosus (SLE). More recently, newly identified noncoding rnas (ncrnas), including long noncoding rnas (lncrnas) and circular rnas (circrnas), are also believed to be involved in the etiology of AIDs while the initial factor behind those epigenetic alterations can be diverse from metabolism to microbiota. Update and comprehensive insights into epigenetics in AIDs can help us understand the pathogenesis and further orchestrate it to benefit patients in the future. Therefore, we reviewed the latest epigenetic findings in SLE, rheumatoid arthritis (RA), Type 1 diabetes (T1D), systemic sclerosis (SSc) primarily from cellular levels.

关键词： Autoimmune disease Epigenetics Lupus Rheumatoid arthritis DNA methylation Histone modification noncoding rna

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An Evaluation of Function of Multicopy noncoding rnas in Mammals Using ENCODE/FANTOM Data and Comparative Genomics

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MOLECULAR BIOLOGY AND EVOLUTION 2018年第6期35卷 1451-1462页

作者： Hoeppner, Marc P. Denisenko, Elena Gardner, Paul P. Schmeier, Sebastian Poole, Anthony M. Christian Albrechts Univ Kiel Inst Clin Mol Biol Kiel Germany Massey Univ Inst Nat & Math Sci Auckland New Zealand Univ Canterbury Sch Biol Sci Biomol Interact Ctr Christchurch New Zealand Univ Auckland Bioinformat Inst Sch Biol Sci Auckland New Zealand

Mammalian diversification has coincided with a rapid proliferation of various types of noncoding rnas, including members of both snrnas and snornas. The significance of this expansion however remains obscure. While some ncrna copy-number expansions have been linked to functionally tractable effects, such events may equally likely be neutral, perhaps as a result of random retrotransposition. Hindering progress in our understanding of such observations is the difficulty in establishing function for the diverse features that have been identified in our own genome. Projects such as ENCODE and FANTOM have revealed a hidden world of genomic expression patterns, as well as a host of other potential indicators of biological function. However, such projects have been criticized, particularly from practitioners in the field of molecular evolution, where many suspect these data provide limited insight into biological function. The molecular evolution community has largely taken a skeptical view, thus it is important to establish tests of function. We use a range of data, including data drawn from ENCODE and FANTOM, to examine the case for function for the recent copy number expansion inmammals of six evolutionarily ancient rna families involved in splicing and rrna maturation. We use several criteria to assess evidence for function: conservation of sequence and structure, genomic synteny, evidence for transposition, and evidence for species-specific expression. Applying these criteria, we find that only aminority of loci show strong evidence for function and that, for the majority, we cannot reject the null hypothesis of no function.

关键词： evolution noncoding rna bioinformatics

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The Role of noncoding rnas in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

Craniomaxillofacial Research & Innovation

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Craniomaxillofacial Research & Innovation 2021年 6卷

作者： Paras Ahmad Martin J. Stoddart Elena Della Bella AO Research Institute Davos Davos Platz Switzerland AO Research Institute Davos Davos Platz SwitzerlandDepartment of Oral and Maxillofacial Surgery Faculty of Medicine Medical Center-University of Freiburg University of Freiburg Freiburg im Breisgau Germany

Chronic inflammatory diseases, including periodontitis, are the most common causes of bone tissue destruction. Periodontitis often leads to loss of connective tissue homeostasis and reduced alveolar bone levels. Human periodontal ligament stem cells (PDLSCs), a population of multipotent stem cells derived from periodontal ligament tissues, are considered as candidate cells for the regeneration of alveolar bone and periodontal tissues. Periodontitis impairs the osteogenic differentiation of human PDLSCs. noncoding rnas (ncrnas), including long noncoding rna (lncrna), microrna (mirna), and circular rna (circrna), have been proposed as vital regulators influencing several differentiation processes including bone regeneration. Still, the molecular mechanisms of ncrnas regulating osteogenic differentiation of human PDLSCs remain poorly understood. Exploring the influence of ncrnas in the process of osteogenic differentiation of human PDLSCs may provide novel therapeutic strategies for tissue regeneration as the regeneration of the lost periodontium is the ultimate goal of periodontal therapy.

关键词： PDLSC periodontitis noncoding rna mirna lncrna circrna

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Serum Expression of Seven Micrornas in Chronic Lymphocytic Leukemia Patients

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JOUrnaL OF BLOOD MEDICINE 2020年 11卷 97-102页

作者： Farzadfard, Ehsan Kalantari, Tahereh Tamaddon, Gholamhossein Shiraz Univ Med Sci Sch Paramed Sci Shiraz Iran Shiraz Univ Med Sci Sch Paramed Sci Dept Med Biotechnol Shiraz Iran Shiraz Univ Med Sci Sch Paramed Sci Dept Clin Lab Sci Shiraz Iran Shiraz Univ Med Sci Sci & Technol Res Ctr Sch Paramed Sci Diagnost Lab Shiraz Iran

Purpose: Micrornas are small single-strand noncoding rnas that can be deregulated in a variety of cancers. Over the past few years, multiple markers have been discovered in chronic lymphocytic leukemia (CLL). Among these, mirnas seem to have important roles in the pathogenesis of CLL. The development and validation of mirna-expression patterns as bio-markers should have a significant impact in cancer diagnosis, therapeutic success, and increasing the life expectancy of patients. In this study, to specify the utility of circulatory mirna expression as noninvasive and useful biomarkers for CLL, we analyzed the dysregulation of seven mirnas: miR30d, miR25-3p, miR19a-3p, miR133b, miR451a, miR145, and miR144 in CLL-patient sera. Methods: Thirty untreated patients with flow-cytometry confirmation of CLL were chosen. Serum samples were collected from 30 newly diagnosed CLL patients. Fifteen healthy samples were taken for comparison as controls. rnawas extracted using Trizol. rna from CLL patient specimens was compared to controls with real-time PCR. Results: Seven mirnas were differently expressed between CLL and normal specimens using the comparative 2(-Delta Delta Ct) method. mirnas 133b, 25-3p, 451a, 145, 19a-3p, and 144 were overexpressed in sera obtained from CLL patients, and mirna-30d was under-expressed in patient samples. Among these seven mirnas, miR19a-3p and miR25-3p showed the most deregulation in CLL patients. Conclusion: Real-time PCR is an applied means to perform high-throughput investigation of serum-rna samples. We assessed the expression of seven mirnas in CLL patients by this method. The results demonstrated that the use of mirna-expression profiling may have an impressive role in the diagnosis of CLL. In addition, mirna 19a-3p and 25-3p are known oncogenes with therapeutic and potential biomarkers.

关键词： chronic lymphocytic leukemia circulatory micrornas noncoding rna real-time PCR

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