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作者： Mara Mancini Anna Maria Lena Eleonora Candi

In situ hybridization (ISH) and fluorescence in situ hybridization (FISH) techniques enable us to detect the expression of a specific rna in fixed cells or tissue sections. Here, we describe in detail two procedures adjusted to reveal specifically lncrnas in normal human keratinocytes and in skin tissue samples. Examples of the results obtained by the two different approaches are also shown. less

关键词： FISH In Situ Hybridization Freeze Fracture Technique Tissue Fixation Cell And Tissue Culture Saccharomyces cerevisiae Skin Keratinocytes noncoding rna Long non coding rna Anti-DIG

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Alternative 3′-end processing of long noncoding rna initiates construction of nuclear paraspeckles

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EMBO JOUrnaL 2012年第20期31卷 4020-4034页

作者： Naganuma, Takao Nakagawa, Shinichi Tanigawa, Akie Sasaki, Yasnory F. Goshima, Naoki Hirose, Tetsuro Natl Inst Adv Ind Sci & Technol Biomed Informat Res Ctr Funct RNom Team Tokyo 1350064 Japan RIKEN Adv Sci Inst RNA Biol Lab Wako Saitama Japan Natl Inst Adv Ind Sci & Technol Biomed Informat Res Ctr Biol Syst Control Team Tokyo 1350064 Japan

Paraspeckles are unique subnuclear structures built around a specific long noncoding rna, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative rna-binding domains. Subsequent rnai analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. The EMBO Journal (2012) 31, 4020-4034. doi:10.1038/emboj.2012.251;Published online 7 September 2012

关键词： alternative rna processing noncoding rna nuclear body rna-binding proteins

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The role of Mediator in small and long noncoding rna production in Arabidopsis thaliana

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EMBO JOUrnaL 2011年第5期30卷 814-822页

作者： Kim, Yun Ju Zheng, Binglian Yu, Yu Won, So Youn Mo, Beixin Chen, Xuemei Univ Calif Riverside Dept Bot & Plant Sci Inst Integrat Genome Biol Riverside CA 92521 USA Shenzhen Univ Coll Life Sci Shenzhen Peoples R China

Mediator is a conserved multi-subunit complex known to promote the transcription of protein-coding genes by rna polymerase II (Pol II) in eukaryotes. It has been increasingly realized that Pol II transcribes a large number of intergenic loci to generate noncoding rnas, but the role of Mediator in Pol II-mediated noncoding rna production has been largely unexplored. The role of Mediator in noncoding rna production in plants is particularly intriguing given that plants have evolved from Pol II two additional polymerases, Pol IV and Pol V, to specialize in noncoding rna production and transcriptional gene silencing at heterochromatic loci. Here, we show that Mediator is required for microrna (mirna) biogenesis by recruiting Pol II to promoters of mirna genes. We also show that several well-characterized heterochromatic loci are de-repressed in Mediator mutants and that Mediator promotes Pol II-mediated production of long noncoding scaffold rnas, which serve to recruit Pol V to these loci. This study expands the function of Mediator to include Pol II-mediated intergenic transcription and implicates a role of Mediator in genome stability. The EMBO Journal (2011) 30, 814-822. doi: 10.1038/emboj.2011.3;Published online 21 January 2011

关键词： Mediator microrna noncoding rna Pol II sirna

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Linc00659, a long noncoding rna, acts as novel oncogene in regulating cancer cell growth in colorectal cancer

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MOLECULAR CANCER 2018年第20180326期17卷 72-72页

作者： Tsai, Kuo-Wang Lo, Yi-Hao Liu, Hsuan Yeh, Chung-Yu Chen, You-Zuo Hsu, Chao-Wen Chen, Wei-Shone Wang, Jui-Ho Kaohsiung Vet Gen Hosp Dept Med Educ & Res Kaohsiung 813 Taiwan Natl Sun Yat Sen Univ Inst Biomed Sci Kaohsiung Taiwan Natl Pingtung Univ Educ Dept Biol Chem Pingtung Taiwan Kaohsiung Armed Forces Gen Hosp Dept Family Med Zuoying Branch Kaohsiung Taiwan Chang Gung Univ Dept Biochem Coll Med Taoyuan Taiwan Chang Gung Mem Hosp Div Colon & Rectal Surg Dept Surg Linkou Taiwan Kaohsiung Vet Gen Hosp Div Colorectal Surg Dept Surg Kaohsiung 813 Taiwan Natl Yang Ming Univ Sch Med Taipei Taiwan Vet Gen Hosp Dept Surg Taipei Taiwan

Background: Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related death worldwide. In patients with CRC, metastasis is a crucial problem that leads to treatment failure and is the primary cause of the lethality of colon cancer. Long noncoding rnas (lncrnas) have recently emerged as critical molecules in the development, cell growth, apoptosis, and metastasis of CRC. Method: We investigated the transcriptome profiles of human lncrnas in the primary tumor tissues and in the corresponding normal mucosa of two patients with CRC by using a microarray approach. The expression levels of lncrnas were verified in colon cancer by real-time PCR. Using bioinformatics approach to illustrate putative biological function of Linc00659 in colon cancer. The effects of Linc00659 on cell growth, proliferation, cell cycle and apoptosis were studies by in vitro assays. Results: Our data revealed that compared with adjacent normal tissues, 201 lncrnas were deregulated (fold change >= 4 or <= 0.25) in CRC tissues. Among them, the expression levels of Linc00659 were significantly increased in colon cancer, and high expression levels were correlated with poor survival in patients with CRC. Bioinformatics analysis results indicated that Linc00659 was significantly coexpressed with cycle-related genes in CRC. Linc00659 expression knockdown could significantly suppress colon cancer cell growth by impairing cell cycle progression. In addition, our results showed that Linc00659 expression knockdown could accelerate cell apoptosis in colon cancer cells treated with chemotherapy drugs. Meanwhile, our results also demonstrated that silencing of Linc00659 expression leads to cell growth inhibition and induced apoptosis, possibly by suppressing PI3K-AKT signaling in colon cancer. Conclusion: Linc00659 is a novel oncogenic lncrna involved in colon cancer cell growth by modulating the cell cycle. Our findings give an insight into lncrna regulation and provide an applicat

关键词： Colorectal carcinoma lncrna noncoding rna

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Methods to Study Long noncoding rna Expression and Dynamics in Zebrafish Using rna Sequencing

Methods to Study Long Noncoding RNA Expression and Dynamics ...

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作者： Samatha Mathew Ambily Sivadas Paras Sehgal Kriti Kaushik Shamsudheen K. Vellarikkal Vinod Scaria Sridhar Sivasubbu

Long noncoding rnas (lncrnas) belong to a class of rna transcripts that do not have the potential to code for proteins. Lncrnas were largely discovered in the transcriptomes of human and several model organisms, using next-generation sequencing (NGS) approaches, which have enabled a comprehensive genome scale annotation of transcripts. Lncrnas are known to have dynamic expression status and have the potential to orchestrate gene regulation at the epigenetic, transcriptional, and posttranscriptional levels. Here we describe the experimental methods involved in the discovery of lncrnas from the transcriptome of a popular model organism zebrafish (Danio rerio). A structured and well-designed computational analysis pipeline subsequent to the rna sequencing can be instrumental in revealing the diversity of the lncrna transcripts. We describe one such computational pipeline used for the discovery of novel lncrna transcripts in zebrafish. We also detail the validation of the putative novel lncrna transcripts using qualitative and quantitative assays in zebrafish. less

关键词： rna-seq rna Extraction Agarose Gel Electrophoresis Library Preparation rna Purification Sequence Generation Statistical Calculation Cufflinks Real-time PCR In Situ Hybridization Antibody-based Screening PCR Restriction Enzyme Digestion In Vitro Transcription Spectroscopy TruSeq DNA Ligation Trimmomatic TopHat Ensembl bedtools Danio rerio Transcriptome noncoding rna Long noncoding rna Anti-DIG

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Genome-Wide Analysis of Long noncoding rna Turnover

Genome-Wide Analysis of Long Noncoding RNA Turnover

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作者： Hidenori Tani Naoto Imamachi Rena Mizutani Katsutoshi Imamura Yeondae Kwon Satoru Miyazaki Sho Maekawa Yutaka Suzuki Nobuyoshi Akimitsu

Genome-wide analysis for determining rna turnover is an advanced method in rna biology that examines the specific half-life of nuclear noncoding rna (ncrna). In particular, a pulse-labeling method using uridine analogs enables the determination of rna stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous rnas in mammalian cells with 5′-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled rnas using deep sequencing. The method is called BrU immunoprecipitation chase assay (BRIC) or BRIC through deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq. less

关键词： Deep Sequencing Metabolic Labeling Pulse Labeling Immunoprecipitation In Vitro Transcription Gel Electrophoresis Bowtie Cufflinks Cuffdiff PCR BRIC-seq Data analysis TruSeq Python DNA Ligation Bos taurus Homo sapiens Mus musculus Gallus gallus Caenorhabditis elegans Drosophila melanogaster Danio rerio Arabidopsis thaliana rna degradation noncoding rna rna turnover 5′-bromo-uridine Anti-BrdU

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Identification of Long noncoding rna-Protein Interactions Through In Vitro rna Pull-Down Assay with Plant Nuclear Extracts

Identification of Long Noncoding RNA-Protein Interactions Th...

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作者： Jun Sung Seo Nam-Hai Chua

Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding rna (lncrna). Many lncrnas function by interacting with proteins and forming regulatory complexes. rna-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncrnas is the first step to deciphering lncrna functions. Here, we describe an rna-protein pull-down assay, which enables the identification of proteins that interact with an rna under study. As an example, we describe pull-down of proteins interacting with lncrna ELENA1, which promotes the enrichment of MED19a on PR1 promoter to activate PR1 expression. less

关键词： In Vitro Transcription Western Blot Protein Extraction Pull-down Assay PCR Sonication Homogenization Bradford Protein Assay PAGE rna pull-down Arabidopsis Oryctolagus cuniculus Biotin noncoding rna rna-protein interactions ELENA1 MED19a Anti-GFP Anti-rabbit

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Targeting noncoding rna Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgrna–ncrna Expression Constructs

Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-...

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作者： David M. Shechner

CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding rna (ncrna) domains to any genomic site, by directly fusing these domains to the Cas9 sgrna cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncrnas, or for utilizing artificial ncrna and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgrna–ncrna fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncrna “cargo” domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgrna–ncrna expression constructs. I detail strategies for choosing expression systems and sgrna insertion topologies, for assaying the incorporation of sgrna–ncrna fusions into functional Cas9 complexes, and for surveying the activities of ncrna domains at targeted genomic loci. This establishes an initial set of “best practices” for the design and implementation of CRISPR-Display experiments. less

关键词： CRISPR rna Secondary Structure Prediction Gibson Assembly Enzymatic Cleavage Sanger Sequencing Cross-linking rna Immunoprecipitation (RIP) Transient Transfection Cell And Tissue Culture Cell Lysis Sonication rna Extraction Real-time PCR Quantitative Reverse Transcription PCR Luciferase Reporter Assay FISH Recombinant Protein Expression Confocal Microscopy Molecular Cloning DNA Ligation Data analysis Pull-down Assay HEK293T lncrna noncoding rna CRISPR-Display Extended sgrna rna Aptamer rna Devices lncrna reconstitution Anti-FLAG Anti-rabbit

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A novel, noncoding-rna-mediated, post-transcriptional mechanism of anti-Mullerian hormone regulation by the H19/let-7 axis

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BIOLOGY OF REPRODUCTION 2019年第1期100卷 101-111页

作者： Qin, Chunrong Xia, Xi Fan, Yanhong Jiang, Ying Chen, Yong Zhang, Na Uslu, Bahar Johnson, Joshua Kallen, Amanda N. Hosp Southern Med Univ Affiliated Shenzhen City Matern & Child Healthcar Ctr Reprod Med Shenzhen Peoples R China Peking Univ Shenzhen Hosp Dept Reprod Med Shenzhen Guangdong Peoples R China Beijing Univ Hosp 3 Dept Obstet & Gynecol Beijing Peoples R China Zhejiang Univ Sch Med Womens Hosp Dept Obstet Hangzhou Zhejiang Peoples R China Fujian Med Univ Sch Basic Med Sci Dept Human Anat & Histol & Embryol Fuzhou Fujian Peoples R China Univ Connecticut Hlth Ctr Dept Genet & Dev Biol Farmington CT USA Quinnipiac Univ Frank H Netter Sch Med North Haven CT USA Univ Colorado Denver AMC Dept Obstet & Gynecol Div Reprod Sci Aurora CO USA Yale Sch Med Dept Obstet Div Reprod Endocrinol & Infertil Gynecol & Reprod Sci New Haven CT USA

In reproductive age women, the pool of primordial follicles is continuously depleted through the process of cyclic recruitment. Anti-Mullerian hormone (AMH) both inhibits the initial recruitment of primordial follicles into the growing pool and modulates the sensitivity of growing follicles to follicle stimulating hormone. Thus, AMH may be an important modulator of female infertility and ovarian reserve;however, the mechanisms regulating AMH remain unclear. To evaluate AMH levels in the absence of H19 lncrna, H19 knockout (H19KO) mice were evaluated for analysis of ovarian AMH gene expression, protein production, and reproductive function, including assessment of follicle numbers and litter size analysis. To further investigate regulation of AMH by the H19/let-7 axis, let-7 binding sites on AMH were predicted, and in vitro studies of the effect of H19 knockdown/overexpression with let-7 rescue were performed. Lastly, response to superovulation was assessed via oocyte counts and estradiol measurements. The H19KO mouse demonstrates subfertility and accelerated follicular recruitment with increased spontaneous development of secondary, preantral, and antral follicles. Ovaries of H19KO mice have decreased AMH mrna and protein, and AMH mrna has a functional let-7 binding site, suggesting a plausible ncrna-mediated mechanism for AMH regulation by H19/let-7. Lastly, in the absence of H19, superovulation results in higher estradiol and more oocytes, suggesting that H19 functions to limit the number of follicles that mature, produce estradiol, and ovulate. Thus, AMH's inhibitory actions are regulated at least in part by H19, likely via let-7, marking this ncrna pair as important regulators of the establishment and maintenance of the follicular pool. The long noncoding rna H19 regulates AMH via let-7.

关键词： AMH ovarian reserve H19 let-7 noncoding rna long noncoding rna microrna mirna lncrna infertility

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noncoding-rna mediated high expression of zinc finger protein 268 suppresses clear cell renal cell carcinoma progression by promoting apoptosis and regulating immune cell infiltration

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BIOENGINEERED 2022年第4期13卷 10467-10481页

作者： Wang, Keyi Gu, Yongzhe Ni, Jinliang Zhang, Houliang Wang, Yidi Zhang, Yifan Sun, Xianchao Xu, Tianyuan Mao, Weipu Peng, Bo Tongji Univ Shanghai Peoples Hosp 10 Sch Med Dept Urol Shanghai Zha Bei Qu Peoples R China Tongji Univ Shanghai Peoples Hosp 10 Sch Med Dept Neurol Shanghai Zha Bei Qu Peoples R China Anhui Med Univ Shanghai Clin Coll Hefei Anhui Peoples R China Southeast Univ Dept Urol Affiliated Zhongda Hosp Nanjing 210009 Jiangsu Peoples R China

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignant kidney tumors with a poor prognosis. Accumulating evidence proves that zinc finger protein 268 (ZNF268) is associated with tumor progression, but the detailed regulatory functions of ZNF268 in ccRCC require further exploration. Thus, here we aim to characterize the role of ZNF268 in ccRCC. The clinical significance of ZNF268 was evaluated using The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. Subsequently, tumor-infiltrating immune cells, as well as upstream noncoding rnas (ncrnas) related to the tumor-suppressing function of ZNF268, were identified by in silico analyses. The expression of ZNF268 was significantly decreased in ccRCC samples compared with adjacent normal tissues. In addition, ZNF268 expression was negatively correlated with tumor progression and positively correlated with overall and disease-specific survival. TCGA and GTEx databases proved the potential tumor-suppressing function, which was measured both in vitro and in vivo after ZNF268 over-expression. Overexpression of ZNF268 effectively inhibited the proliferation, migration, invasion and promotied apoptosis of the Caki-1. The level of ZNF268 was positively related to the immune cell infiltration in the tumor. Moreover, we determined that the AC093157.1/miR-27a-3p axis can potentially regulate ZNF268 function in ccRCC. Our work describes a novel ncrna-mediated ZNF268 function in ccRCC. ZNF268 acts as a tumor suppressor, and it is associated with apoptosis and immune cell infiltration in ccRCC.

关键词： ZNF268 kidney renal clear cell carcinoma prognosis immune infiltration noncoding rna

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