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作者： Eleonora D’Ambra Mariangela Morlando

Circular rnas (circrnas) are covalently closed transcripts generated by back-splicing reaction. The lack of free ends endows these rna molecules with high stability thus allowing them to accumulate in tissues and body fluids. They are widely expressed in most organisms, are modulated during development and display tissue-specific expression, resulting particularly enriched in the nervous system. Deregulation of circrna expression has also been associated with several pathological conditions including neurological diseases and cancer. Here we present a Northern blot procedure that allows the analysis of the expression of bona fide circrnas through the use of a digoxigenin-labeled rna probe and the immunodetection of the signals. less

关键词： rna Electrophoresis Immunoassay Agarose Gel Electrophoresis Northern Blot PCR Electrophoresis rna probes Northern Blots Digoxigenin noncoding rna circrna

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Long-Read Sequencing - A Powerful Toll in Viral Transcriptome Research

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TRENDS IN MICROBIOLOGY 2019年第7期27卷 578-592页

作者： Boldogkoi, Zsolt Moldovan, Norbert Balazs, Zsolt Snyder, Michael Tombacz, Ddra Univ Szeged Fac Med Dept Med Biol Szeged Hungary Stanford Univ Sch Med Dept Genet 300 Pasteur Dr Stanford CA 94305 USA

Long-read sequencing (LRS) has become increasingly popular due to its strengths in de novo assembly and in resolving complex DNA regions as well as in determining full-length rna molecules. Two important LRS technologies have been developed during the past few years, including single-molecule, real-time sequencing by Pacific Biosciences, and nanopore sequencing by Oxford Nanopore Technologies. Although current LRS methods produce lower coverage, and are more error prone than short-read sequencing, these methods continue to be superior in identifying transcript isoforms including multispliced rnas and transcript-length variants as well as overlapping transcripts and alternative polycistronic rna molecules. Viruses have small, compact genomes and therefore these organisms are ideal subjects for transcriptome analysis with the relatively low-throughput LRS techniques. Recent LRS studies have multiplied the number of previously known transcripts and have revealed complex networks of transcriptional overlaps in the examined viruses.

关键词： DNA replication replication origin nanopore sequencing Pacific Biosciences rna-Seq noncoding rna splicing

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Biology of rna Surveillance in Development and Disease

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TRENDS IN CELL BIOLOGY 2019年第5期29卷 428-445页

作者： Laffleur, Brice Basu, Uttiya Columbia Univ Dept Microbiol & Immunol Vagelos Coll Phys & Surg New York NY 10032 USA

The 'rna world', in which rna molecules stored information and acquired enzymatic properties, has been proposed to have preceded organism life. rna is now recognized for its central role in biology, with accumulating evidence implicating coding and noncoding (nc)rnas in myriad mechanisms regulating cellular physiology and disequilibrium in transcriptomes resulting in pathological conditions. Nascently synthesized rnas are subjected to stringent regulation by sophisticated rna surveillance pathways. In this review, we integrate these pathways from a developmental viewpoint, proposing rna surveillance as the convergence of mechanisms that ensure the exact titration of rna molecules in a spatiotemporally controlled manner, leading to development without the onset of pathological conditions, including cancer.

关键词： rna surveillance rna processing noncoding rna differentiation development developmental disease

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Caste-specific microrna expression in termites: insights into soldier differentiation

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INSECT MOLECULAR BIOLOGY 2019年第1期28卷 86-98页

作者： Matsunami, M. Nozawa, M. Suzuki, R. Toga, K. Masuoka, Y. Yamaguchi, K. Maekawa, K. Shigenobu, S. Miura, T. Hokkaido Univ Grad Sch Environm Sci Lab Ecol Genet Sapporo Hokkaido Japan Tokyo Metropolitan Univ Dept Biol Sci Hachioji Tokyo Japan Univ Toyama Grad Sch Sci & Engn Toyama Japan Natl Inst Basic Biol Funct Genom Facil Okazaki Aichi Japan Univ Ryukyus Grad Sch Med 207 Nishihara Cho Nishihara Okinawa 9030215 Japan Nihon Univ Coll Humanities & Sci Dept Biosci Tokyo Japan Natl Agr & Food Res Org Inst Agrobiol Sci Tsukuba Ibaraki Japan Univ Tokyo Misaki Marine Biol Stn Miura Kanagawa Japan

Eusocial insects have polyphenic caste systems in which each caste exhibits characteristic morphology and behaviour. In insects, caste systems arose independently in different lineages, such as Isoptera and Hymenoptera. Although partial molecular mechanisms for the development of eusociality in termites have been clarified by the functional analysis of genes and hormones, the contribution of micrornas (mirnas) to caste differentiation is unknown. To understand the role of mirnas in termite caste polyphenism, we performed small rna sequencing in a subterranean termite (Reticulitermes speratus) and identified the mirnas that were specifically expressed in the soldier and worker castes. Of the 550 mirnas annotated in the R. speratus genome, 74 were conserved in insects and 174 were conserved in other termite species. We found that eight mirnas (mir-1, mir-125, mir-133, mir-2765, mir-87a and three termite-specific mirnas) are differentially expressed (DE) in soldiers and workers of R. speratus. This differential expression was experimentally verified for five mirnas by real-time quantitative PCR. Further, four of the eight DE mirnas in soldier and worker termite castes were also differentially expressed in hymenopteran castes. The finding that Isoptera and Hymenoptera shared several DE mirnas amongst castes suggests that these mirnas evolved independently in these phylogenetically distinct lineages.

关键词： noncoding rna microrna termite phenotypic plasticity gene expression eusociality.

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Angiogenin generates specific stress-induced trna halves and is not involved in tRF-3?mediated gene silencing

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JOUrnaL OF BIOLOGICAL CHEMISTRY 2019年第45期294卷 16930-16941页

作者： Su, Zhangli Kuscu, Canan Malik, Asrar Shibata, Etsuko Dutta, Anindya Univ Virginia Dept Biochem & Mol Genet Charlottesville VA 22901 USA Univ Tennessee Transplant Res Inst Dept Surg Hlth Sci Ctr Memphis TN 38163 USA

trna fragments (tRFs) and trna halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more. Overexpressed angiogenin (ANG) cleaves trna anticodons and produces trna halves similar to those produced in response to stress. However, it is not clear whether endogenous ANG is essential for producing the stress-induced trna halves. It is also not clear whether smaller tRFs are generated from the trna halves. Here, using global short rna-Seq approach, we found that ANG overexpression selectively cleaves a subset of trnas, including trna(Glu), trna(Gly), trna(Lys), trna(Val), trna(His), trna(Asp), and trna(SeC) to produce trna halves and tRF-5s that are 26?30 bases long. Surprisingly, ANG knockout revealed that the majority of stress-induced trna halves, except for the 5? half from trna(HisGTG) and the 3? half from trna(AspGTC), are ANG independent, suggesting there are other rnases that produce trna halves. We also found that the 17?25 bases?long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from trna halves. Consistent with this, ANG knockout did not decrease tRF-3 levels or gene-silencing activity. We conclude that ANG cleaves specific trnas and is not the only rnase that creates trna halves and that the shorter tRFs are not generated from the trna halves or from independent trna cleavage by ANG.

关键词： rna metabolism rna processing transfer rna (trna) ribonuclease stress angiogenin gene regulation noncoding rna small rna trna fragment

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An Easy, Cost-Effective, and Scalable Method to Deplete Human Ribosomal rna for rna-seq

CURRENT PROTOCOLS

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CURRENT PROTOCOLS 2021年第6期1卷 e176页

作者： Baldwin, Amber Morris, Adam R. Mukherjee, Neelanjan Univ Colorado Anschutz Sch Med Aurora CO 80309 USA

rna sequencing (rna-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total rna is ribosomal rna (rrna). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger rna (mrna) and long noncoding rnas (lncrna), it is essential to minimize the rrna being sequenced. Although commercial methods exist to deplete rrna from total rna samples before sequencing, they are expensive and require specific amounts of input rna, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rrna depletion protocol using rnase H and DNA oligonucleotides complementary to human rrna transcripts. This protocol includes guidelines for DNA oligo preparation, rna:DNA hybridization, rnase H cleavage and rna cleanup, and benchmarking of rrna depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rrna depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input rna amounts. (c) 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rrna transcripts from human total rna Support Protocol: Preparation of the rrna depletion DNA oligonucleotide pool

关键词： mrna noncoding rna ribosomal rna rna-seq

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Ro60 and Y rnas: structure, functions, and roles in autoimmunity

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CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 2019年第2期54卷 133-152页

作者： Boccitto, Marco Wolin, Sandra L. NCI RNA Biol Lab Ctr Canc Res Frederick MD 21702 USA

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved rna-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding rnas called Y rnas. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y rnas on its outer surface. In addition to Y rnas, Ro60 binds misfolded and aberrant noncoding rnas in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding rnas in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3 to 5 exoribonucleases to assist structured rna degradation. Studies of Y rnas have revealed that these rnas regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other rnas to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y rnas in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.

关键词： Ro60 Y rna Rsr autoantigen noncoding rna rna degradation

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Utility Of Plasma circBNC2 As A Diagnostic Biomarker In Epithelial Ovarian Cancer

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ONCOTARGETS AND THERAPY 2019年 12卷 9715-9723页

作者： Hu, Yingchao Zhu, Yapei Zhang, Wen Lang, Jinghe Ning, Li Chinese Acad Med Sci Peking Union Med Coll Hosp Dept Obstet & Gynecol Beijing 100730 Peoples R China Peking Union Med Coll Beijing 100730 Peoples R China Chinese Acad Med Sci Canc Hosp Dept Gynecol Oncol Natl Canc Ctr Beijing 100021 Peoples R China Peking Union Med Coll Beijing 100021 Peoples R China

Background: Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer death in women. Due to a lacking of early detection method, its five-year survival rate is only 30%. Nevertheless, novel biomarkers for diagnosis remain to be discovered. The potential of microrna signatures in the diagnosis of EOC has been especially described in recent years. In our previous experiments, we identified that circBNC2 was downregulated in EOC specimens, and was associated with advanced tumor stage and lymph node metastasis (LNM) by performing circrna-sequencing analysis. The aim of this study was to explore the diagnostic value of circBNC2 in patients with epithelial ovarian cancer (EOC). Methods: Plasma from 249 age and menopause-matched women (83 with EOC;83 with benign ovarian cyst;83 were healthy volunteers) was collected prior to surgery. CircBNC2 was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) were analyzed using enzyme-linked immunosorbent assay (ELISA). Receiver operating curve (ROC), the area under the curve (AUC), sensitivity and specificity were estimated. Results: CircBNC2 was downregulated in EOC and had higher ROC AUC in comparing EOC to benign (ROC AUC 0.879, sensitivity 96.4%, specificity 80.7%) or healthy (ROC AUC 0.923, sensitivity 95.2%, specificity 85.5%) cohorts than HE4 (ROC AUC: 0.742, benign cohort;0.779, healthy cohort) and CA125 (ROC AUC: 0.373, benign cohort;0.713, healthy cohort). Early stage EOC vs benign (ROC AUC 0.864, sensitivity 92.0%, specificity 80.7%) and healthy (ROC AUC 0.908, sensitivity 92.0%, specificity 85.5%) cohorts could be significantly separated by circBNC2. CircBNC2 performed alike in pre- and postmenopausal women, within EOC compared to the benign or healthy cohort. Conclusion: CircBNC2 is downregulated in EOC (both in tissue and plasma samples) and might present promising novel biomarker for EOC. Further studies

关键词： epithelial ovarian cancer circular rna noncoding rna diagnosis biomarker

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Identification of differentially expressed circular rnas in chemoresistant colorectal cancer

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EPIGENOMICS 2019年第8期11卷 875-884页

作者： Abu, Nadiah Hon, Kha Wai Jeyaraman, Shivapriya Yahaya, Azyani Abdullah, Nik M. A. Mustangin, Muaatamarulain Sulaiman, Siti A. Jamal, Rahman Ab-Mutalib, Nurul-S Univ Kebangsaan Malaysia UKM Med Mol Biol Inst UMBI Kuala Lumpur 56000 Malaysia Univ Kebangsaan Malaysia Dept Pathol Fac Med UKM Med Ctr Kuala Lumpur 56000 Malaysia Univ Kebangsaan Malaysia Dept Oncol & Radiotherapy Fac Med UKM Med Ctr Kuala Lumpur 56000 Malaysia

Chemoresistance in colorectal cancer (CRC) has become a burden in treating the disease effectively. Circular rnas (circrnas) are a type of noncoding rna that were found to be important in cellular homeostasis. The involvement of circrnas in relation to chemoresistance in other types of cancers has also been reported. This study aims to identify the differentially expressed circrnas between chemoresistant and chemosensitive CRC cells. Materials & methods: We developed a chemoresistant cell line model and profiled the circrnas via microarray. We further validated the expression of two circrnas in 25 formalin-fixed paraffin-embedded (FFPE) tissue specimens (13 nonresponders and 12 responders) via quantitative polymerase chain reaction (qPCR). Results & conclusion: We found that there were 773 upregulated and 732 downregulated circrnas between the chemoresistant and chemosensitive HCT-116 cells. We found that hsa_circ_32883 could be a promising biotarget.

关键词： circrna circrna-mirna-m rna interaction colorectal cancer drug resistance 5-fluorouracil FOL-FOX HCT116 microarray noncoding rna oxaliplatin

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A novel assessing system for predicting the prognosis of gastric cancer

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EPIGENOMICS 2019年第11期11卷 1251-1266页

作者： Lin, Siheng Zhou, Rui Zeng, Dongqiang Wu, Jiani Wu, Jianhua Zhang, Jingwen Sun, Huiying Zhu, Shaowei Shi, Min Bin, Jianping Liao, Yulin Liao, Wangjun Southern Med Univ Nanfang Hosp Dept Oncol Guangzhou Guangdong Peoples R China Southern Med Univ Nanfang Hosp Dept Med Ultrason Guangzhou Guangdong Peoples R China Southern Med Univ Nanfang Hosp Dept Cardiol State Key Lab Organ Failure Res Guangzhou Guangdong Peoples R China

Aim: To develop novel diagnostic tools that can predict the prognosis of gastric cancer. Material & methods: Using rna expression data from The Cancer Genome Atlas and Gene Expression Omnibus, we established protein-coding rnas-noncoding rnas-tumor microenvironment type (PNM) scores, which contain signatures of tumor protein coding genes (P), tumor noncoding genes (N) and immune/stroma cells in tumor microenvironment (M) to predict the prognosis of gastric cancer. Results & conclusion: Based on PNM scores, gastric cancer patients were divided into three subgroups and Kaplan-Meier survival curves revealed significant differences among the subgroups (p < 0.001). Our study showed that the PNM scores could be used as a robust predicting tool for the prognosis of gastric cancer.

关键词： gastric cancer immunotherapy noncoding rna prognosis tumor microenvironment

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