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Yao Z, Jin H, Li C, Ma W, Zhang W, Lin Y. Knockdown of Dcr1 and Dcr2 limits the lethal effect of C-factor in Chilo suppressalis. ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY 2023; 113:e22004. [PMID: 36780173 DOI: 10.1002/arch.22004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Revised: 01/13/2023] [Accepted: 01/31/2023] [Indexed: 06/18/2023]
Abstract
Dicer is a highly conserved ribonuclease in evolution. It belongs to the RNase III family and can specifically recognize and cleave double-stranded RNA (dsRNA). In this study, the genome and transcriptome of Chilo suppressalis were analyzed, and it was found that there were two members in the Dicer family, named Dcr1 and Dcr2. The dsRNAs of Dcr1 and Dcr2 genes were synthesized and fed to C. suppressalis larvae. The C-factor of C. suppressalis was selected as the marker gene. The results showed that both Dcr1 and Dcr2 genes were significantly knocked down. The larval mortality was significantly reduced by 43.50% (p < 0.05) after feeding on dsC-factor and dsDcr1. The transcription levels of C-factor genes were significantly increased by 33.95% (p < 0.05) and 32.94% (p < 0.05) when the larvae fed with dsDcr2 + dsC-factor for 72 h and 96 h, respectively. Furthermore, the mortality was significantly decreased by 79% (p < 0.05) after feeding dsC-factor and dsDcr2. These findings imply that Dcr1 can decrease the lethal effect of C-factor gene but cannot affect its RNAi efficiency and Dcr2 can decrease the lethal effect of C-factor gene by inhibiting RNAi efficiency.
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Affiliation(s)
- Zhuotian Yao
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Huihui Jin
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Changyan Li
- National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, Hubei, China
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Weihua Ma
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
- National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Wei Zhang
- National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, Hubei, China
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Yongjun Lin
- National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, Hubei, China
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
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Tsouris A, Schacherer J, Ishchuk OP. RNA Interference (RNAi ) as a Tool for High-Resolution Phenotypic Screening of the Pathogenic Yeast Candida glabrata. Methods Mol Biol 2022; 2477:313-330. [PMID: 35524125 DOI: 10.1007/978-1-0716-2257-5_18] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
After its discovery RNA interference (RNAi) has become a powerful tool to study gene functions in different organisms. RNAi has been applied at genome-wide scale and can be nowadays performed using high-throughput automated systems (robotics). The simplest RNAi process requires the expression of two genes (Dicer and Argonaute) to function. To initiate the silencing, constructs generating either double-strand RNA or antisense RNA are required. Recently, RNAi was reconstituted by expressing Saccharomyces castellii genes in the human pathogenic yeast Candida glabrata and was used to identify new genes related to the virulence of this pathogen.In this chapter, we describe a method to make the C. glabrata pathogenic yeast competent for RNAi and to use RNA silencing as a tool for low- or high-resolution phenotypic screening in this species.
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Affiliation(s)
- Andreas Tsouris
- Université de Strasbourg, CNRS, GMGM UMR 7156, Strasbourg, France
| | - Joseph Schacherer
- Université de Strasbourg, CNRS, GMGM UMR 7156, Strasbourg, France
- Institut Universitaire de France (IUF), Paris, France
| | - Olena P Ishchuk
- Department of Biology and Biological Engineering, Systems and Synthetic Biology, Chalmers University of Technology, Gothenburg, Sweden.
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Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors. Pharmaceutics 2021; 13:pharmaceutics13050676. [PMID: 34066833 PMCID: PMC8151203 DOI: 10.3390/pharmaceutics13050676] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 04/30/2021] [Accepted: 05/04/2021] [Indexed: 11/17/2022] Open
Abstract
Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition.
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Aryal NK, Pant V, Wasylishen AR, Rimel BJ, Baseler L, El-Naggar AK, Mutch DG, Goodfellow PJ, Arur S, Lozano G. Dicer1 Phosphomimetic Promotes Tumor Progression and Dissemination. Cancer Res 2019; 79:2662-2668. [PMID: 30914430 DOI: 10.1158/0008-5472.can-18-2460] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Revised: 10/23/2018] [Accepted: 03/22/2019] [Indexed: 11/16/2022]
Abstract
Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/- ). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion. SIGNIFICANCE: This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.
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Affiliation(s)
- Neeraj K Aryal
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas.,Genes and Development Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas
| | - Vinod Pant
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Amanda R Wasylishen
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Bobbie J Rimel
- Division of Gynecologic Oncology, Cedars Sinai Medical Center, Los Angeles, California
| | - Laura Baseler
- Department of Veterinary Medicine and Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Adel K El-Naggar
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - David G Mutch
- Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Washington University School of Medicine and Siteman Cancer Center, St. Louis, Missouri
| | - Paul J Goodfellow
- Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, The Ohio State University and James Comprehensive Cancer Center, Columbus, Ohio
| | - Swathi Arur
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas. .,Genes and Development Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas
| | - Guillermina Lozano
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas. .,Genes and Development Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas
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5
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Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo. Proc Natl Acad Sci U S A 2018; 116:960-969. [PMID: 30593561 DOI: 10.1073/pnas.1814377116] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1 S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.
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Tietze S, Schau I, Michen S, Ennen F, Janke A, Schackert G, Aigner A, Appelhans D, Temme A. A Poly(Propyleneimine) Dendrimer-Based Polyplex-System for Single-Chain Antibody-Mediated Targeted Delivery and Cellular Uptake of SiRNA. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2017; 13:1700072. [PMID: 28544767 DOI: 10.1002/smll.201700072] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Revised: 04/13/2017] [Indexed: 06/07/2023]
Abstract
Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.
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Affiliation(s)
- Stefanie Tietze
- Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany
| | - Isabell Schau
- Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany
| | - Susanne Michen
- Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany
| | - Franka Ennen
- Leibniz Institute of Polymer Research Dresden, Hohe Straße 6, 01069, Dresden, Germany
| | - Andreas Janke
- Leibniz Institute of Polymer Research Dresden, Hohe Straße 6, 01069, Dresden, Germany
| | - Gabriele Schackert
- Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany
- German Cancer Consortium (DKTK), Partner Site Dresden German Cancer Research Center (DKFZ) Heidelberg, German and National Center for Tumor Diseases (NCT), 01307, Dresden, Germany
| | - Achim Aigner
- Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University Medicine Leipzig, Härtelstraße 16-18, 04107, Leipzig, Germany
| | - Dietmar Appelhans
- Leibniz Institute of Polymer Research Dresden, Hohe Straße 6, 01069, Dresden, Germany
| | - Achim Temme
- Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany
- German Cancer Consortium (DKTK), Partner Site Dresden German Cancer Research Center (DKFZ) Heidelberg, German and National Center for Tumor Diseases (NCT), 01307, Dresden, Germany
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Paces J, Nic M, Novotny T, Svoboda P. Literature review of baseline information to support the risk assessment of RNAi‐based GM plants. ACTA ACUST UNITED AC 2017. [PMCID: PMC7163844 DOI: 10.2903/sp.efsa.2017.en-1246] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Jan Paces
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
| | | | | | - Petr Svoboda
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
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8
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Fu Y, Zhang J, Shi Z, Wang G, Li W, Jia L. A key gene of the small RNA pathway in the flounder, Paralichthys olivaceus: identification and functional characterization of dicer. FISH PHYSIOLOGY AND BIOCHEMISTRY 2015; 41:1221-1231. [PMID: 26045159 DOI: 10.1007/s10695-015-0081-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 05/25/2015] [Indexed: 06/04/2023]
Abstract
Dicer is critical for producing mature microRNAs (miRNAs) from precursor molecules and small interfering RNAs and plays an important role in controlling development and metabolism. In the present study, we cloned the flounder dicer gene, which is 6585 nucleotides (nt), including a 5'-untranslated region (UTR) of 231 nt, a 3'-UTR of 663 nt and an open reading frame of 5691 nt encoding a polypeptide of 1897 amino acids, and analyzed the conservation and expression pattern of dicer. The tissue distribution analysis indicated that dicer is abundantly expressed in the brain, heart, liver, spleen, stomach, kidney, gill, muscle, intestine and gonad of adult fish. Temporal expression analysis indicated that dicer mRNA is highly expressed during the embryonic and early larval stages, and exhibits low expression during the metamorphic stages. Treatment with thyroid hormone (TH) or thiourea indirectly or directly up-regulated dicer mRNA levels at 17 and 23 dph, whereas treatment with TH down-regulated dicer mRNA levels at 36 dph. The dicer-specific siRNA significantly down-regulated dicer mRNA and pol-let-7d levels, while pol-let-7d precursor levels were not differentially changed compared with the control (NC). These results demonstrated that dicer plays a key role in development and metabolism through the production of mature miRNAs, providing basic information for further studies concerning the role of dicer in Paralichthys olivaceus development.
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Affiliation(s)
- Yuanshuai Fu
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China
| | - Junling Zhang
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China
| | - Zhiyi Shi
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China.
| | - Guyue Wang
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China
| | - Wejuan Li
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China
| | - Liang Jia
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999, Hu-Cheng-Huan Road, Lingang New City, Shangai, 201306, China
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Abstract
microRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They emerged more than 20 years ago as key posttranscriptional regulators of gene expression. The regulatory role of these small RNA molecules has recently begun to be explored in the human reproductive system. microRNAs have been shown to play an important role in control of reproductive functions, especially in the processes of oocyte maturation, folliculogenesis, corpus luteum function, implantation, and early embryonic development. Knockout of Dicer, the cytoplasmic enzyme that cleaves the pre-miRNA to its mature form, results in postimplantation embryonic lethality in several animal models, attributing to these small RNA vital functions in reproduction and development. Another intriguing characteristic of microRNAs is their presence in body fluids in a remarkably stable form that is protected from endogenous RNase activity. In this chapter we will describe the current knowledge on microRNAs, specifically relating to human gonadal cells. We will focus on their role in the ovarian physiologic process and ovulation dysfunction, regulation of spermatogenesis and male fertility, and putative involvement in human normal and aberrant trophoblast differentiation and invasion through the process of placentation.
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Thongthae N, Payungporn S, Poovorawan Y, T-Thienprasert NP. A rational study for identification of highly effective siRNAs against hepatitis B virus. Exp Mol Pathol 2014; 97:120-127. [PMID: 24953337 DOI: 10.1016/j.yexmp.2014.06.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2014] [Revised: 06/07/2014] [Accepted: 06/18/2014] [Indexed: 02/07/2023]
Abstract
RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317-1337, 1357-1377 and 1644-1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.
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Affiliation(s)
- Nuttkawee Thongthae
- Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
| | - Sunchai Payungporn
- Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Yong Poovorawan
- Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
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Imbar T, Eisenberg I. Regulatory role of microRNAs in ovarian function. Fertil Steril 2014; 101:1524-30. [DOI: 10.1016/j.fertnstert.2014.04.024] [Citation(s) in RCA: 88] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Revised: 04/13/2014] [Accepted: 04/15/2014] [Indexed: 01/08/2023]
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12
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Hu S, Cao W, Yang M, Liu H, Li L, Wang J. Molecular characterization, tissue distribution, and expression of two ovarian Dicer isoforms during follicle development in goose (Anser cygnoides). Comp Biochem Physiol B Biochem Mol Biol 2014; 170:33-41. [DOI: 10.1016/j.cbpb.2014.01.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Revised: 01/09/2014] [Accepted: 01/13/2014] [Indexed: 12/24/2022]
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13
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Klein S, Lee H, Ghahremani S, Kempert P, Ischander M, Teitell MA, Nelson SF, Martinez-Agosto JA. Expanding the phenotype of mutations in DICER1: mosaic missense mutations in the RNase IIIb domain of DICER1 cause GLOW syndrome. J Med Genet 2014; 51:294-302. [PMID: 24676357 DOI: 10.1136/jmedgenet-2013-101943] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
BACKGROUND Constitutional DICER1 mutations have been associated with pleuropulmonary blastoma, cystic nephroma, Sertoli-Leydig tumours and multinodular goitres, while somatic DICER1 mutations have been reported in additional tumour types. Here we report a novel syndrome termed GLOW, an acronym for its core phenotypic findings, which include Global developmental delay, Lung cysts, Overgrowth and Wilms tumour caused by mutations in the RNase IIIb domain of DICER1. METHODS AND RESULTS We performed whole exome sequencing on peripheral mononuclear blood cells of an affected proband and identified a de novo missense mutation in the RNase IIIb domain of DICER1. We confirmed an additional de novo missense mutation in the same domain of an unrelated case by Sanger sequencing. These missense mutations in the RNase IIIb domain of DICER1 are suspected to affect one of four metal binding sites located within this domain. Pyrosequencing was used to determine the relative abundance of mutant alleles in various tissue types. The relative mutation abundance is highest in Wilms tumour and unaffected kidney samples when compared with blood, confirming that the mutation is mosaic. Finally, we performed bioinformatic analysis of microRNAs expressed in murine cells carrying specific Dicer1 RNase IIIb domain metal binding site-associated mutations. We have identified a subset of 3p microRNAs that are overexpressed whose target genes are over-represented in mTOR, MAPK and TGF-β signalling pathways. CONCLUSIONS We propose that mutations affecting the metal binding sites of the DICER1 RNase IIIb domain alter the balance of 3p and 5p microRNAs leading to deregulation of these growth signalling pathways, causing a novel human overgrowth syndrome.
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Affiliation(s)
- Steven Klein
- Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California, USA
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14
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Loss of functional Dicer in mouse radial glia cell-autonomously prolongs cortical neurogenesis. Dev Biol 2013; 382:530-7. [PMID: 24012747 PMCID: PMC3793872 DOI: 10.1016/j.ydbio.2013.08.023] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Revised: 08/07/2013] [Accepted: 08/26/2013] [Indexed: 11/23/2022]
Abstract
Radial glia of the mouse cerebral cortex emerge from neuroepithelial stem cells around embryonic day 11 and produce excitatory cortical neurons until a few days before birth. The molecular mechanisms that regulate the end of cortical neurogenesis remain largely unknown. Here we investigated if the Dicer-dependent microRNA (miRNA) pathway is involved. By electroporating a cre-recombinase expression vector into the cortex of E13.5 embryos carrying a conditional allele of Dicer1, we induced mosaic recombination causing Dicer1 deletion and reporter activation in a subset of radial glia. We analysed the long-term fates of their progeny. We found that mutant radial glia produced abnormally large numbers of Cux1-positive neurons, many of which populated the superficial cortical layers. Injections of the S-phase marker bromodeoxyuridine between postnatal days 3 and 14 showed that much of this population was generated postnatally. Our findings suggest a role for Dicer-dependent processes in limiting the timespan of cortical neurogenesis.
Analysis of radial glia development in the absence of functional Dicer. Loss of Dicer prolongs mouse cortical neurogenesis postnatally. We found no defects in the onset of gliogenesis.
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15
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Ma E, Zhou K, Kidwell MA, Doudna JA. Coordinated activities of human dicer domains in regulatory RNA processing. J Mol Biol 2012; 422:466-76. [PMID: 22727743 DOI: 10.1016/j.jmb.2012.06.009] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2012] [Revised: 05/31/2012] [Accepted: 06/01/2012] [Indexed: 12/22/2022]
Abstract
The conserved ribonuclease Dicer generates microRNAs and short-interfering RNAs that guide gene silencing in eukaryotes. The specific contributions of human Dicer's structural domains to RNA product length and substrate preference are incompletely understood, due in part to the difficulties of Dicer purification. Here, we show that active forms of human Dicer can be assembled from recombinant polypeptides expressed in bacteria. Using this system, we find that three distinct modes of RNA recognition give rise to Dicer's fidelity and product length specificity. The first involves anchoring one end of a double-stranded RNA helix within the PAZ domain, which can assemble in trans with Dicer's catalytic domains to reconstitute an accurate but non-substrate-selective dicing activity. The second entails nonspecific RNA binding by the double-stranded RNA binding domain, an interaction that is essential for substrate recruitment in the absence of the PAZ domain. The third mode of recognition involves hairpin RNA loop recognition by the helicase domain, which ensures efficient processing of specific substrates. These results reveal distinct interactions of each Dicer domain with different RNA structural features and provide a facile system for investigating the molecular mechanisms of human microRNA biogenesis.
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Affiliation(s)
- Enbo Ma
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
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16
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Motl JA, Chalker DL. Zygotic expression of the double-stranded RNA binding motif protein Drb2p is required for DNA elimination in the ciliate Tetrahymena thermophila. EUKARYOTIC CELL 2011; 10:1648-59. [PMID: 22021239 PMCID: PMC3232721 DOI: 10.1128/ec.05216-11] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/19/2011] [Accepted: 10/13/2011] [Indexed: 11/20/2022]
Abstract
Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome.
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Affiliation(s)
- Jason A. Motl
- Department of Biology, Washington University in St. Louis, Campus Box 1137, One Brookings Dr., St. Louis, Missouri 63130-4899
| | - Douglas L. Chalker
- Department of Biology, Washington University in St. Louis, Campus Box 1137, One Brookings Dr., St. Louis, Missouri 63130-4899
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Nowakowski TJ, Mysiak KS, Pratt T, Price DJ. Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon. PLoS One 2011; 6:e23013. [PMID: 21826226 PMCID: PMC3149632 DOI: 10.1371/journal.pone.0023013] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2011] [Accepted: 07/11/2011] [Indexed: 11/18/2022] Open
Abstract
Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.
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Affiliation(s)
- Tomasz Jan Nowakowski
- Developmental Biology Laboratory, Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.
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18
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Scott MS, Ono M. From snoRNA to miRNA: Dual function regulatory non-coding RNAs. Biochimie 2011; 93:1987-92. [PMID: 21664409 PMCID: PMC3476530 DOI: 10.1016/j.biochi.2011.05.026] [Citation(s) in RCA: 178] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2011] [Accepted: 05/19/2011] [Indexed: 11/03/2022]
Abstract
Small nucleolar RNAs (snoRNAs) are an ancient class of small non-coding RNAs present in all eukaryotes and a subset of archaea that carry out a fundamental role in the modification and processing of ribosomal RNA. In recent years, however, a large proportion of snoRNAs have been found to be further processed into smaller molecules, some of which display different functionality. In parallel, several studies have uncovered extensive similarities between snoRNAs and other types of small non-coding RNAs, and in particular microRNAs. Here, we explore the extent of the relationship between these types of non-coding RNA and the possible underlying evolutionary forces that shaped this subset of the current non-coding RNA landscape.
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Affiliation(s)
- Michelle S Scott
- Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
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19
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Tan GS, Garchow BG, Liu X, Metzler D, Kiriakidou M. Clarifying mammalian RISC assembly in vitro. BMC Mol Biol 2011; 12:19. [PMID: 21529364 PMCID: PMC3112105 DOI: 10.1186/1471-2199-12-19] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2010] [Accepted: 04/29/2011] [Indexed: 01/08/2023] Open
Abstract
Background Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active in vitro and constitute non-canonical RISCs. Results The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates in vitro RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate in vitro pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes. Conclusions Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.
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Affiliation(s)
- Grace S Tan
- Department of Medicine, Division of Rheumatology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
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20
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Smalheiser NR, Lugli G, Thimmapuram J, Cook EH, Larson J. Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training. RNA (NEW YORK, N.Y.) 2011; 17:166-181. [PMID: 21045079 PMCID: PMC3004058 DOI: 10.1261/rna.2123811] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2010] [Accepted: 09/30/2010] [Indexed: 05/29/2023]
Abstract
We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.
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Affiliation(s)
- Neil R Smalheiser
- Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
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Ni M, Shu W, Bo X, Wang S, Li S. Correlation between sequence conservation and structural thermodynamics of microRNA precursors from human, mouse, and chicken genomes. BMC Evol Biol 2010; 10:329. [PMID: 20977776 PMCID: PMC2984420 DOI: 10.1186/1471-2148-10-329] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2010] [Accepted: 10/27/2010] [Indexed: 01/22/2023] Open
Abstract
Background Previous studies have shown that microRNA precursors (pre-miRNAs) have considerably more stable secondary structures than other native RNAs (tRNA, rRNA, and mRNA) and artificial RNA sequences. However, pre-miRNAs with ultra stable secondary structures have not been investigated. It is not known if there is a tendency in pre-miRNA sequences towards or against ultra stable structures? Furthermore, the relationship between the structural thermodynamic stability of pre-miRNA and their evolution remains unclear. Results We investigated the correlation between pre-miRNA sequence conservation and structural stability as measured by adjusted minimum folding free energies in pre-miRNAs isolated from human, mouse, and chicken. The analysis revealed that conserved and non-conserved pre-miRNA sequences had structures with similar average stabilities. However, the relatively ultra stable and unstable pre-miRNAs were more likely to be non-conserved than pre-miRNAs with moderate stability. Non-conserved pre-miRNAs had more G+C than A+U nucleotides, while conserved pre-miRNAs contained more A+U nucleotides. Notably, the U content of conserved pre-miRNAs was especially higher than that of non-conserved pre-miRNAs. Further investigations showed that conserved and non-conserved pre-miRNAs exhibited different structural element features, even though they had comparable levels of stability. Conclusions We proposed that there is a correlation between structural thermodynamic stability and sequence conservation for pre-miRNAs from human, mouse, and chicken genomes. Our analyses suggested that pre-miRNAs with relatively ultra stable or unstable structures were less favoured by natural selection than those with moderately stable structures. Comparison of nucleotide compositions between non-conserved and conserved pre-miRNAs indicated the importance of U nucleotides in the pre-miRNA evolutionary process. Several characteristic structural elements were also detected in conserved pre-miRNAs.
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Affiliation(s)
- Ming Ni
- Center for Bioinformatics, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China
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Huang CCJ, Yao HHC. Inactivation of Dicer1 in Steroidogenic factor 1-positive cells reveals tissue-specific requirement for Dicer1 in adrenal, testis, and ovary. BMC DEVELOPMENTAL BIOLOGY 2010; 10:66. [PMID: 20540774 PMCID: PMC2897782 DOI: 10.1186/1471-213x-10-66] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/20/2009] [Accepted: 06/11/2010] [Indexed: 11/22/2022]
Abstract
Background The synthesis of microRNA (miRNA) is a multi-step process that requires the action of the ribonuclease Dicer1. Dicer1 is responsible for the final processing of miRNA and has been implicated in cellular processes such as proliferation, apoptosis, and differentiation. Mouse embryos lacking Dicer1 die in early embryogenesis. In this study, we investigated whether Dicer1 is required for development of adrenal, testis, and ovary in mouse embryos. Results To target Dicer1 deletion specifically in developing adrenals and gonads, we used Steroidogenic factor 1-cre (Sf1/Cre) line in which Cre recombinase is active in the progenitor cells of adrenals and gonads. Lack of Dicer1 in the SF1-positive cells did not affect formation and early differentiation of the adrenals and gonads. However, increasing numbers of apoptotic cells were first detected in the Dicer1 knockout adrenal cortex at 18.5 days post coitum (dpc), followed by apoptosis of somatic cells and germ cells in the testis at postnatal day 0. Affected adrenal and testes underwent complete degeneration 48 hrs after the onset of apoptosis. However, ovaries were not affected at least until postnatal day 5, when the animals died due to adrenal insufficiency. Conclusions Dicer1 is dispensable for formation and differentiation of fetal tissues derived from the SF1-positive adrenogonadal primordium. Dicer1 is essential for maintaining cell survival in adrenal and testis; however, development of the ovary from fetal stages to postnatal day 5 does not require the presence of Dicer1. Our results reveal a tissue-specific requirement of Dicer1 and microRNAs. Future research is needed to understand how the tissue-specific role of Dicer1 is established.
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Affiliation(s)
- Chen-Che J Huang
- Department of Veterinary Biosciences, University of Illinois, Urbana, IL, USA
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MacFarlane LA, Gu Y, Casson AG, Murphy PR. Regulation of fibroblast growth factor-2 by an endogenous antisense RNA and by argonaute-2. Mol Endocrinol 2010; 24:800-12. [PMID: 20197313 DOI: 10.1210/me.2009-0367] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
We have previously reported that elevated fibroblast growth factor-2 (FGF-2) expression is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer and that these risks are reduced in tumors coexpressing an endogenous antisense (FGF-AS) RNA. In the present study, we examined the role of the endogenous FGF-AS transcript in the regulation of FGF-2 expression in the human lung adenocarcinoma cell line Seg-1. FGF-2 and FGF-AS were temporally and spatially colocalized in the cytoplasm of individual cells, and knockdown of either FGF-2 or FGF-AS by target-specific siRNAs resulted in dose-dependent up-regulation of the complementary transcript and its encoded protein product. Using a luciferase reporter system, we show that these effects are mediated by interaction of the endogenous antisense RNA with the 3'-untranslated region of the FGF-2 mRNA. Deletion mapping identified a 392-nucleotide sequence in the 5823-nucleotide FGF-2 untranslated tail that is targeted by FGF-AS. Small interfering RNA-mediated knockdown of either FGF-AS or FGF-2 significantly increased the stability of the complementary partner mRNA, demonstrating that these mRNAs are mutually regulatory. Knockdown of FGF-AS also resulted in reduced expression of argonaute-2 (AGO-2) and a number of other elements of the endogenous micro-RNA/RNA interference pathways. Conversely, small interfering RNA-mediated knockdown of AGO-2 significantly increased the stability of the FGF-2 mRNA transcript and the steady-state levels of both FGF-2 mRNA and protein, suggesting a role for AGO-2 in the regulation of FGF-2 expression.
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Affiliation(s)
- Leigh-Ann MacFarlane
- Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
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Luense LJ, Carletti MZ, Christenson LK. Role of Dicer in female fertility. Trends Endocrinol Metab 2009; 20:265-72. [PMID: 19646895 PMCID: PMC3121329 DOI: 10.1016/j.tem.2009.05.001] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2008] [Revised: 05/08/2009] [Accepted: 05/08/2009] [Indexed: 12/21/2022]
Abstract
Dicer is an RNAse III endonuclease that is essential for the biogenesis of microRNAs and small interfering RNAs. These small RNAs post-transcriptionally regulate mRNA gene expression through several mechanisms to affect key cellular events including proliferation, differentiation and apoptosis. Recently, the role of Dicer function in female reproductive tissues has begun to be elucidated through the use of knockout mouse models. Loss of Dicer within ovarian granulosa cells, luteal tissue, oocyte, oviduct and, potentially, the uterus renders females infertile. This review discusses these early studies and other data describing the current understanding of microRNAs and small interfering RNAs in female reproduction.
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Affiliation(s)
- Lacey J Luense
- Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA
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25
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Zou J, Chang M, Nie P, Secombes CJ. Origin and evolution of the RIG-I like RNA helicase gene family. BMC Evol Biol 2009; 9:85. [PMID: 19400936 PMCID: PMC2686710 DOI: 10.1186/1471-2148-9-85] [Citation(s) in RCA: 191] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2008] [Accepted: 04/28/2009] [Indexed: 12/24/2022] Open
Abstract
BACKGROUND The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. RESULTS The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. CONCLUSION The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.
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Affiliation(s)
- Jun Zou
- Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, UK.
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Jin L, Kryukov K, Suzuki Y, Imanishi T, Ikeo K, Gojobori T. The evolutionary study of small RNA-directed gene silencing pathways by investigating RNase III enzymes. Gene 2009; 435:1-8. [PMID: 19393176 DOI: 10.1016/j.gene.2008.12.022] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2007] [Revised: 11/29/2008] [Accepted: 12/23/2008] [Indexed: 11/28/2022]
Abstract
The RNA-mediated gene silencing pathways are evolutionarily conserved processes. They highlight a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defense. Recently three distinct small RNA-directed silencing pathways are observed, such as the destruction of mRNA via siRNA, inhibition of mRNA translation via miRNA, and epigenetic gene silencing via siRNA. It was also found that in these pathways, the members of ribonuclease III family play important roles in diverse RNA maturation and decay. Here we investigated the evolution of RNase III nucleases, Dicer as representative, to further figure out the evolutionary relationship of these three gene silencing pathways. With the advantage of using genomic sequences as the subject in homolog search, in un-annotated genomic regions, we were able to detect possible candidates for 3 functional domains and genes of dicer and drosha. Moreover, we found that prokaryotes including eubacteria and archaea lack completely the PAZ domain of Dicer. These results show the taxonomic-dependent evolution of the RNA-mediated gene silencing pathways.
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Affiliation(s)
- Lihua Jin
- Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Yata, Mishima, Shizuoka, Japan
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Soifer HS, Sano M, Sakurai K, Chomchan P, Saetrom P, Sherman MA, Collingwood MA, Behlke MA, Rossi JJ. A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs. Nucleic Acids Res 2008; 36:6511-22. [PMID: 18927112 PMCID: PMC2582626 DOI: 10.1093/nar/gkn687] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.
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Affiliation(s)
- Harris S Soifer
- Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
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Liu X, Yang J, Shang F, Hong C, Guo W, Wang B, Zheng Q. Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA. Mol Biol Rep 2008; 36:1345-52. [PMID: 18636235 DOI: 10.1007/s11033-008-9318-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2008] [Accepted: 07/07/2008] [Indexed: 11/26/2022]
Abstract
GIRK4 has been shown to be a subunit of I(KACh), and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, I(KACh) densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased I(KACh) densities. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.
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Affiliation(s)
- Xiongtao Liu
- Department of Cardiology, Affiliated Tangdu Hospital of the Fourth Military Medical University, Xi'an, 710038, People's Republic of China
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Gewirtz AM. On future's doorstep: RNA interference and the pharmacopeia of tomorrow. J Clin Invest 2008; 117:3612-4. [PMID: 18060018 DOI: 10.1172/jci34274] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Small molecules and antibodies have revolutionized the treatment of malignant diseases and appear promising for the treatment of many others. Nonetheless, there are many candidate therapeutic targets that are not amenable to attack by the current generation of targeted therapies, and in a small but growing number of patients, resistance to initially successful treatments evolves. This Review Series on the medicinal promise of posttranscriptional gene silencing with small interfering RNA and other molecules capable of inducing RNA interference (RNAi) is motivated by the hypothesis that effectors of RNAi can be developed into effective drugs for treating malignancies as well as many other types of disease. As this Review Series points out, there is still much to do, but many in the field now hope that the time has finally arrived when "antisense" therapies will finally come of age and fulfill their promise as the magic bullets of the 21st century.
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Affiliation(s)
- Alan M Gewirtz
- Division of Hematology/Oncology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6061, USA.
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Abstract
RNA interference is involved in many aspects of cell biology, and the recent identification of germ-cell specific small RNAs has led to speculation that RNAi might also be involved in gametogenesis. Work in yeast indicates that RNAi is involved in establishing and maintaining heterochromatin at centromeres, an important component of yeast and mammalian meiosis. Here we review developments in the field of RNAi and relate these to possible roles in mammalian gametogenesis.
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Affiliation(s)
- Rebecca J Holmes
- Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
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Chiosea S, Jelezcova E, Chandran U, Luo J, Mantha G, Sobol RW, Dacic S. Overexpression of Dicer in precursor lesions of lung adenocarcinoma. Cancer Res 2007; 67:2345-50. [PMID: 17332367 DOI: 10.1158/0008-5472.can-06-3533] [Citation(s) in RCA: 188] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Differential microRNA (miR) expression is described in non-small cell lung carcinoma. miR biogenesis requires a set of proteins collectively referred to as the miR machinery. In the proposed multistep carcinogenesis model, peripheral adenocarcinoma of the lung develops from noninvasive precursor lesions known as atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC). The gene array analysis of BAC and adenocarcinoma showed a transient up-regulation of Dicer (a key effector protein for small interfering RNA and miR function) and PACT along with down-regulation of most genes encoding miR machinery proteins. Immunohistochemically, Dicer was up-regulated in AAH and BAC and down-regulated in areas of invasion and in advanced adenocarcinoma. A fraction of adenocarcinomas lose Dicer as a result of deletions at the Dicer locus. Expanded immunohistochemical and Western blot analysis showed higher Dicer level in squamous cell carcinoma (SCC) of the lung when compared with adenocarcinoma. Other proteins of the RNA-induced silencing complex (RISC; SND1, PACT, and FXR1) were also present at higher levels in a SCC cell line when compared with an adenocarcinoma cell line. In conclusion, the stoichiometry of miR machinery and RISC depends on histologic subtype of lung carcinoma, varies along the AAH-BAC-adenocarcinoma sequence, and might explain the observed abnormal miR profile in lung cancer. The status of the endogenous miR machinery in various histologic subtypes and stages of lung cancer may help to predict the toxicity of and susceptibility to future RNA interference-based therapy.
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Affiliation(s)
- Simion Chiosea
- Department of Pathology, University of Pittsburgh Medical Center Presbyterian, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
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Bähr M, Lingor P. Brain repair: Experimental treatment strategies, neuroprotective and repair strategies in the lesioned adult CNS. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2007; 557:148-63. [PMID: 16955709 DOI: 10.1007/0-387-30128-3_9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Affiliation(s)
- Mathias Bähr
- Department of Neurology, University of Göttingen, Germany
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Abstract
RNA-mediated interference (RNAi) is a recently discovered process by which dsRNA is able to silence specific gene functions. Although initially described in plants, nematodes and Drosophila, the process is currently considered to be an evolutionarily conserved process that is present in the entire eukaryotic kingdom in which its original function was as a defense mechanism against viruses and foreign nucleic acids. Similarly to the silencing of genes by RNAi, viral functions can be also silenced by the same mechanism, through the introduction of specific dsRNA molecules into cells, where they are targeted to essential genes or directly to the viral genome in case RNA viruses, thus arresting viral replication. Since the pioneering work of Elbashir and coworkers, who identified RNAi activity in mammalian cells, many publications have described the inhibition of viruses belonging to most if not all viral families, by targeting and silencing diverse viral genes as well as cell genes that are essential for virus replication. Moreover, virus expression vectors were developed and used as vehicles with which to deliver siRNAs into cells. This review will describe the use of RNAi to inhibit virus replication directly, as well as through the silencing of the appropriate cell functions.
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Affiliation(s)
- Yehuda Stram
- Virology Division, Kimron Veterinary institute, 12, 50250, Beit-Dagan, Israel.
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Zhang H, Barnoski BL, Sol-Church K, Stabley DL, Martin-Deleon PA. Murine Spam1 mRNA: involvement of AU-rich elements in the 3'UTR and antisense RNA in its tight post-transcriptional regulation in spermatids. Mol Reprod Dev 2006; 73:247-55. [PMID: 16250006 DOI: 10.1002/mrd.20400] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Sperm adhesion molecule1 (SPAM1), the best characterized hyaluronidase gene, is abundantly expressed in the testis. We attempted to overexpress mouse Spam1 via transgenesis using either the endogenous promoter in a BAC or a heterologous Protamine1 promoter for a Spam1 cDNA transgene. Although transgene-copy numbers ranged from 2 to 15 and transgenic transcripts were expressed, there was a general failure of overexpression of the RNA and protein in the testis of all seven founders. Also, three transgenic lines showed a modest downregulation or co-suppression of the RNA for Spam1 and Hyal5, present on the BAC. We provide evidence for the potential involvement of two co-ordinating post-transcriptional regulatory processes in the failure of overexpression: abundant endogenous antisense RNA and adenosine-uridine (AU)-rich element-mediated regulation of RNA turnover. We demonstrate that AU-rich elements (AREs) in the 3'UTR of mRNAs, well-known to interact with trans-acting proteins to target the RNA for (in)stability, are present in Spam1 RNA and specifically bind to six testicular cytoplasmic proteins. These AU-binding proteins (AUBPs) were virtually absent from the kidney where transcripts are rare, and were shown to interact with the cytoskeleton, which modulates mRNA turnover. In addition to a role in the RNAi pathway, antisense RNA can also modulate ARE-mediated regulation of mRNA by hybridizing to the AREs and specifically silencing their function. This potentially links the two processes in the regulation of Spam1 expression. We hypothesize that testicular Spam1 RNA is regulated post-transcriptionally by cis-acting ARE(s) in the 3'UTR which recognize AUBPs and which are modulated by antisense transcripts.
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Affiliation(s)
- Hong Zhang
- Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
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Murchison EP, Hannon GJ. miRNAs on the move: miRNA biogenesis and the RNAi machinery. Curr Opin Cell Biol 2005; 16:223-9. [PMID: 15145345 DOI: 10.1016/j.ceb.2004.04.003] [Citation(s) in RCA: 279] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Recent advances have led to a more detailed understanding of RNA interference and its role in microRNA biogenesis and function. Primary microRNA transcripts are processed by the RNaseIII nuclease, Drosha, and are exported from the nucleus by Exportin-5. Dicer cleaves microRNAs into their mature forms, which can be incorporated into effector complexes that mediate gene silencing activities. The 3' two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway. MicroRNA functional analyses and genetic and biochemical interrogation of components of the pathway are starting to provide a glimpse at the range of biological processes and phenomena regulated by RNA interference.
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Affiliation(s)
- Elizabeth P Murchison
- Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
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Lugli G, Larson J, Martone ME, Jones Y, Smalheiser NR. Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner. J Neurochem 2005; 94:896-905. [PMID: 16092937 DOI: 10.1111/j.1471-4159.2005.03224.x] [Citation(s) in RCA: 206] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
We have hypothesized that small RNAs may participate in learning and memory mechanisms. Because dendritic spines are important in synaptic plasticity and learning, we asked whether dicer, the rate-limiting enzyme in the formation of small RNAs, is enriched within dendritic spines. In adult mouse brain, dicer and the RNA-induced silencing complex (RISC) component eIF2c were expressed in the somatodendritic compartment of principal neurons and some interneurons in many regions, and dicer was enriched in dendritic spines and postsynaptic densities (PSDs). A portion of dicer and eIF2c were associated with each other and with fragile X mental retardation protein (FMRP), as assessed by co-immunoprecipitation. Calpain I treatment of recombinant dicer or immunopurified brain dicer caused a marked increase in RNAse III activity. Purified PSDs did not exhibit RNAse III activity, but calpain caused release of dicer from PSDs in an enzymatically active form, together with eIF2c. NMDA stimulation of hippocampal slices, or calcium treatment of synaptoneurosomes, caused a 75 kDa dicer fragment to appear in a calpain-dependent manner. The findings support a model whereby acute neuronal stimulation at excitatory synapses increases intracellular calcium, which activates calpain, which liberates dicer and eIF2c bound to PSDs. This supports the hypothesis that dicer could be involved in synaptic plasticity.
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Affiliation(s)
- Giovanni Lugli
- University of Illinois at Chicago, UIC Psychiatric Institute, Chicago, Illinois, USA
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Abstract
The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.
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Affiliation(s)
- R Michael Raab
- Department of Chemical Engineering, Room 56-459, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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Sago N, Omi K, Tamura Y, Kunugi H, Toyo-oka T, Tokunaga K, Hohjoh H. RNAi induction and activation in mammalian muscle cells where Dicer and eIF2C translation initiation factors are barely expressed. Biochem Biophys Res Commun 2004; 319:50-7. [PMID: 15158441 DOI: 10.1016/j.bbrc.2004.04.151] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2004] [Indexed: 10/26/2022]
Abstract
Dicer plays an important role in the course of RNA interference (RNAi), i.e., it digests long double-stranded RNAs into 21-25 nucleotide small-interfering RNA (siRNA) duplexes functioning as sequence-specific RNAi mediators. In this study, we investigated the expression levels of Dicer and eIF2C1 approximately 4, which, like Dicer, appear to participate in mammalian RNAi, in various mouse tissues. Results indicate that the levels of eIF2C1 approximately 4 as well as Dicer are lower in skeletal muscle and heart than in other tissues. To see if RNAi could occur under such a condition with low levels of expression of Dicer and eIF2C1 approximately 4, we examined RNAi activity in mouse skeletal muscle fibers. The results indicate that RNAi can be induced by synthetic siRNA duplexes in muscle fibers. We further examined RNAi activity during myogenic differentiation of mouse C2C12 cells. The data indicate that although the expression levels of Dicer and eIF2C1 approximately 4 decrease during the differentiation, RNAi can be induced in the cells. Altogether, the data presented here suggest that muscle cells retain the ability to induce RNAi, although Dicer and eIF2C1 approximately 4 appear to be barely expressed in them.
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Affiliation(s)
- Noriko Sago
- National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
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Abstract
First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complex that degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins--RNAi technology may eventually make that dream a reality.
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Affiliation(s)
- Ollivier Milhavet
- Laboratory of Neurosciences, National Institute on Aging, Gerontology Research Center, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
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40
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Abstract
Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statistical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5'-anti-sense (AS) terminal base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal stability (Delta0.5 kcal/mol) at the 5'-AS end than nonfunctional duplexes. Average internal stability of siRNA molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.
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Abstract
Elucidation of many disease-related signal transduction and gene expression pathways has provided unparalleled opportunities for the development of targeted therapeutics. The types of molecules in development are increasingly varied and include small-molecule enzyme inhibitors, humanized antibodies to cell surface receptors, and antisense nucleic acids for silencing the expression of specific genes. This Perspective reviews the basis for various antisense strategies for modulating gene expression, including RNA interference, and discusses the prospects for their clinical use.
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Affiliation(s)
- Joanna B Opalinska
- Department of Hematology, Pommeranian Medical Academy, ul. Unii Lubelskiej 1, 71245 Szczecin, Poland.
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42
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Affiliation(s)
- Douglas S Conklin
- Cancer Genome Center, Cold Spring Harbor Laboratory, 500 Sunnyside Blvd, Woodbury, NY 11797, USA.
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43
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Abstract
RNA interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals. RNA interference is part of a sophisticated network of interconnected pathways for cellular defense, RNA surveillance, and development and it may become a powerful tool to manipulate gene expression experimentally. RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop specific dsRNA based gene-silencing therapeutics. Several laboratories have been interested in using RNAi to control viral infection and many reports in Nature and in Cell show that short interfering (si) RNAs can inhibit infection by HIV-1, polio and hepatitis C viruses in a sequence-specific manner. RNA-based strategies for gene inhibition in mammalian cells have recently been described, which offer the promise of antiviral therapy.
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Affiliation(s)
- Quan-Chu Wang
- The Center of Diagnosis and Treatment for Infectious Diseases of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
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Abstract
The dsRNA binding proteins (DRBPs) comprise a growing family of eukaryotic, prokaryotic, and viral-encoded products that share a common evolutionarily conserved motif specifically facilitating interaction with dsRNA. Proteins harboring dsRNA binding domains (DRBDs) have been reported to interact with as little as 11 bp of dsRNA, an event that is independent of nucleotide sequence arrangement. More than 20 DRBPs have been identified and reportedly function in a diverse range of critically important roles in the cell. Examples include the dsRNA-dependent protein kinase PKR that functions in dsRNA signaling and host defense against virus infection and DICER, which is implicated in RNA interference (RNAi) -mediated gene silencing. Other DRBPs such as Staufen, adenosine deaminase acting on RNA (ADAR), and spermatid perinuclear RNA binding protein (SPNR) are known to play essential roles in development, translation, RNA editing, and stability. In many cases, homozygous and even heterozygous disruption of DRBPs in animal models results in embryonic lethality. These results implicate the recognition of dsRNA as an evolutionarily conserved mechanism important in the regulation of gene expression and in host defense and underscore the diversity of essential biological tasks performed by dsRNA-related processes in the cell.
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Affiliation(s)
- Laura R Saunders
- Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida, USA
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Stein P, Svoboda P, Anger M, Schultz RM. RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA (NEW YORK, N.Y.) 2003; 9:187-92. [PMID: 12554861 PMCID: PMC1370384 DOI: 10.1261/rna.2860603] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/25/2002] [Accepted: 10/23/2002] [Indexed: 05/19/2023]
Abstract
Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.
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Affiliation(s)
- Paula Stein
- Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA
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Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 2002; 21:5875-85. [PMID: 12411505 PMCID: PMC131079 DOI: 10.1093/emboj/cdf582] [Citation(s) in RCA: 448] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.
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Affiliation(s)
| | | | - Vincent Brondani
- Friedrich Miescher Institute for Biomedical Research, PO Box 2543, CH-4002 Basel, Switzerland
Present address: Institute of Medical Microbiology, University of Basel, CH-4003 Basel, Switzerland Present address: Novartis Pharma AG, CH-4056 Basel, Switzerland Corresponding author e-mail:
| | - Eric Billy
- Friedrich Miescher Institute for Biomedical Research, PO Box 2543, CH-4002 Basel, Switzerland
Present address: Institute of Medical Microbiology, University of Basel, CH-4003 Basel, Switzerland Present address: Novartis Pharma AG, CH-4056 Basel, Switzerland Corresponding author e-mail:
| | - Witold Filipowicz
- Friedrich Miescher Institute for Biomedical Research, PO Box 2543, CH-4002 Basel, Switzerland
Present address: Institute of Medical Microbiology, University of Basel, CH-4003 Basel, Switzerland Present address: Novartis Pharma AG, CH-4056 Basel, Switzerland Corresponding author e-mail:
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Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Rådmark O. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J 2002; 21:5864-74. [PMID: 12411504 PMCID: PMC131075 DOI: 10.1093/emboj/cdf578] [Citation(s) in RCA: 332] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.
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Affiliation(s)
- Patrick Provost
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
| | - David Dishart
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
| | - Johanne Doucet
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
| | - David Frendewey
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
| | - Bengt Samuelsson
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
| | - Olof Rådmark
- Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada and Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA Corresponding authors e-mail: or
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48
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Fortin KR, Nicholson RH, Nicholson AW. Mouse ribonuclease III. cDNA structure, expression analysis, and chromosomal location. BMC Genomics 2002; 3:26. [PMID: 12191433 PMCID: PMC122089 DOI: 10.1186/1471-2164-3-26] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2002] [Accepted: 08/21/2002] [Indexed: 11/22/2022] Open
Abstract
BACKGROUND Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III. RESULTS The predicted mouse RNase III polypeptide contains 1373 amino acids (approximately 160 kDa). The polypeptide exhibits a single C-terminal dsRNA-binding motif (dsRBM), tandem catalytic domains, a proline-rich region (PRR) and an RS domain. Northern analysis and RT-PCR reveal that the transcript (4487 nt) is expressed in all tissues examined, including extraembryonic tissues and the midgestation embryo. Northern analysis indicates the presence of an additional, shorter form of the transcript in testicular tissue. Fluorescent in situ hybridization demonstrates that the mouse RNase III gene maps to chromosome 15, region B, and that the human RNase III gene maps to a syntenic location on chromosome 5p13-p14. CONCLUSIONS The broad transcript expression pattern indicates a conserved cellular role(s) for mouse RNase III. The putative polypeptide is highly similar to human RNase III (99% amino acid sequence identity for the two catalytic domains and dsRBM), but is distinct from other eukaryotic orthologues, including Dicer, which is involved in RNA interference. The mouse RNase III gene has a chromosomal location distinct from the Dicer gene.
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Affiliation(s)
- Kristine R Fortin
- Department of Biological Sciences, Wayne State University 5047 Gullen Mall, Detroit, MI 48202
| | - Rhonda H Nicholson
- Department of Biological Sciences, Wayne State University 5047 Gullen Mall, Detroit, MI 48202
| | - Allen W Nicholson
- Department of Biological Sciences, Wayne State University 5047 Gullen Mall, Detroit, MI 48202
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49
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Abstract
RNAi is evolving into a powerful tool for manipulating gene expression in mammalian cells with potential utility for investigating gene function, for high-throughput, function-based genetic screens and potentially for development as a therapeutic tool.
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Affiliation(s)
- Patrick J Paddison
- Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA
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