An Overview on Ribonucleases and Their Therapeutic Effects
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Anti-Dicer (SAB4200087)
Anti-Dicer produced in rabbit, affinity isolated antibody Product Number SAB4200087 Product Description Precautions and Disclaimer Anti-Dicer is produced in rabbit using as the For R&D use only. Not for drug, household, or other immunogen a synthetic peptide corresponding to a uses. Please consult the Safety Data Sheet for fragment of human Dicer (Gene ID: 23405) conjugated information regarding hazards and safe handling to KLH. The corresponding sequence is identical in practices. mouse. The antibody is affinity-purified using the immunizing peptide immobilized on agarose. Storage/Stability Store at –20 C. For continuous use, store at 2–8 C for Anti-Dicer recognizes human Dicer. The antibody may up to one month. For extended storage, freeze in be used in several immunochemical techniques working aliquots at –20 C. Repeated freezing and including immunoblotting (218 kDa), immuno- thawing, or storage in “frost-free” freezers, is not precipitation, and immunofluorescence. Detection of recommended. If slight turbidity occurs upon prolonged the Dicer band by immunoblotting is specifically storage, clarify the solution by centrifugation before inhibited with the immunizing peptide. use. Working dilutions should be discarded if not used within 12 hours. Dicer, also known as Dicer1, Endoribonuclease Dicer, Helicase with RNase motif, and HERNA, is a member Product Profile of the RNase III family that catalyzes the first step in the Immunoblotting: a working antibody concentration of RNA interference (RNAi) pathway and initiates 3-6 g/mL is recommended using HeLa cell lysates. formation of the RNA-induced silencing complex (RISC). Dicer processes the dsRNA into small Immunoprecipitation: a working antibody amount of fragments called short interfering RNA (siRNA) or 2.5-5 g is recommended using HeLa cell lysates. -
Enzymes and Rna Complexes
ENZYMES AND RNA COMPLEXES Mediator NMD Exosome NMD TRAMP/NNS Integrator Microprocessor RNA PROCESSING and DECAY machinery: RNases Protein Function Characteristics Exonucleases 5’ 3’ Xrn1 cytoplasmic, mRNA degradation processsive Rat1 nuclear, pre-rRNA, sn/snoRNA, pre-mRNA processing and degradation Rrp17/hNol12 nuclear, pre-rRNA processing Exosome 3’ 5’ multisubunit exo/endo complex subunits organized as in bacterial PNPase Rrp44/Dis3 catalytic subunit Exo/PIN domains, processsive Rrp4, Rrp40 pre-rRNA, sn/snoRNA processing, mRNA degradation Rrp41-43, 45-46 participates in NMD, ARE-dependent, non-stop decay Mtr3, Ski4 Mtr4 nuclear helicase cofactor DEAD box Rrp6 (Rrp47) nuclear exonuclease ( Rrp6 BP, cofactor) RNAse D homolog, processsive Ski2,3,7,8 cytoplasmic exosome cofactors. SKI complex helicase, GTPase Other 3’ 5’ Rex1-4 3’-5’ exonucleases, rRNA, snoRNA, tRNA processing RNase D homolog DXO 3’-5’ exonuclease in addition to decapping mtEXO 3’ 5’ mitochondrial degradosome RNA degradation in yeast Suv3/ Dss1 helicase/ 3’-5’ exonuclease DExH box/ RNase II homolog Deadenylation Ccr4/NOT/Pop2 major deadenylase complex (Ccr, Caf, Pop, Not proteins) Ccr4- Mg2+ dependent endonuclease Pan2p/Pan3 additional deadenylases (poliA tail length) RNase D homolog, poly(A) specific nuclease PARN mammalian deadenylase RNase D homolog, poly(A) specific nuclease Endonucleases RNase III -Rnt1 pre-rRNA, sn/snoRNA processing, mRNA degradation dsRNA specific -Dicer, Drosha siRNA/miRNA biogenesis, functions in RNAi PAZ, RNA BD, RNase III domains Ago2 Slicer -
Ribonuclease A
Chem. Rev. 1998, 98, 1045−1065 1045 Ribonuclease A Ronald T. Raines Departments of Biochemistry and Chemistry, University of WisconsinsMadison, Madison, Wisconsin 53706 Received October 10, 1997 (Revised Manuscript Received January 12, 1998) Contents I. Introduction 1045 II. Heterologous Production 1046 III. Structure 1046 IV. Folding and Stability 1047 A. Disulfide Bond Formation 1047 B. Prolyl Peptide Bond Isomerization 1048 V. RNA Binding 1048 A. Subsites 1048 B. Substrate Specificity 1049 C. One-Dimensional Diffusion 1049 D. Processive Catalysis 1050 VI. Substrates 1050 VII. Inhibitors 1051 Ronald T. Raines was born in 1958 in Montclair, NJ. He received Sc.B. VIII. Reaction Mechanism 1052 degrees in chemistry and biology from the Massachusetts Institute of A. His12 and His119 1053 Technology. At M.I.T., he worked with Christopher T. Walsh to reveal the reaction mechanisms of pyridoxal 5′-phosphate-dependent enzymes. B. Lys41 1054 Raines was a National Institutes of Health predoctoral fellow in the C. Asp121 1055 chemistry department at Harvard University. There, he worked with D. Gln11 1056 Jeremy R. Knowles to elucidate the reaction energetics of triosephosphate IX. Reaction Energetics 1056 isomerase. Raines was a Helen Hay Whitney postdoctoral fellow in the biochemistry and biophysics department at the University of California, A. Transphosphorylation versus Hydrolysis 1056 San Francisco. At U.C.S.F., he worked with William J. Rutter to clone, B. Rate Enhancement 1057 express, and mutate the cDNA that codes for ribonuclease A. Raines X. Ribonuclease S 1058 then joined the faculty of the biochemistry department at the University s A. S-Protein−S-Peptide Interaction 1058 of Wisconin Madison, where he is now associate professor of biochem- istry and chemistry. -
Characterization of the Mammalian RNA Exonuclease 5/NEF-Sp As a Testis-Specific Nuclear 3′′′′′ → 5′′′′′ Exoribonuclease
Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Characterization of the mammalian RNA exonuclease 5/NEF-sp as a testis-specific nuclear 3′′′′′ → 5′′′′′ exoribonuclease SARA SILVA,1,2 DAVID HOMOLKA,1 and RAMESH S. PILLAI1 1Department of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland 2European Molecular Biology Laboratory, Grenoble Outstation, 38042, France ABSTRACT Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3′′′′′ → 5′′′′′ exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2′′′′′-O-methyl modification at the 3′′′′′ end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome. Keywords: NEF-sp; LOC81691; Q96IC2; REXON; RNA exonuclease 5; REXO5; 2610020H08Rik INTRODUCTION clease-mediated processing to create their final 3′ ends: poly(A) tails of most mRNAs or the hairpin structure of Spermatogenesis is the process by which sperm cells are replication-dependent histone mRNAs (Colgan and Manley produced in the male germline. -
Protein Engineering to Exploit and Explore Bovine Secretory Ribonucleases
PROTEIN ENGINEERING TO EXPLOIT A N D EXPLORE BOVINE SECRETORY RIBONUCLEASES by JIN-SOO KIM A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 1994 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to thank Dr. Ronald T. Raines for his advice and support. His scientific insight has been very helpful throughout this work. I would also like to thank the entire Raines group for their friendship and companionship. I am grateful to Dr. J. Soucek and Dr. J. Matousek for their collaboration with us, which has been a valuable part of the BS-RNase research. I thank Dr. M. Karpeisky for suggesting the protein fusion project, and Dr. G. D'Alessio and Dr. L. Mazzarella for providing the coordinates of BS-RNase. I have been generously supported by Steenbock predoctoral fellowship from the Department of Biochemistry. Finally, I thank my parents, who have encouraged (or at least not discouraged) me to pursue a career in science since I was a kid. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission ABSTRACT PROTEIN ENGINEERING TO EXPLOIT AND EXPLORE BOVINE SECRETORY RIBONUCLEASES Jin-Soo Kim Under the supervision of Dr. Ronald T. Raines at the University of Wisconsin-Madison Ribonuclease S-peptide (residues 1-20) and S-protein (residues 21- 124) are the enzymatically inactive products of the limited digestion of bovine pancreatic ribonuclease A (RNase A) by subtilisin. S-Peptide binds S-protein with high affinity to form RNase S, which has full enzymatic activity. -
DICER1 Gene Dicer 1, Ribonuclease III
DICER1 gene dicer 1, ribonuclease III Normal Function The DICER1 gene provides instructions for making a protein that plays a role in regulating the activity (expression) of other genes. The Dicer protein aids in the production of a molecule called microRNA (miRNA). MicroRNAs are short lengths of RNA, a chemical cousin of DNA. Dicer cuts (cleaves) precursor RNA molecules to produce miRNA. MicroRNAs control gene expression by blocking the process of protein production. In the first step of making a protein from a gene, another type of RNA called messenger RNA (mRNA) is formed and acts as the blueprint for protein production. MicroRNAs attach to specific mRNA molecules and stop the process by which protein is made. Sometimes, miRNAs break down the mRNA, which also blocks protein production. Through this role in regulating the expression of genes, Dicer is involved in many processes, including cell growth and division (proliferation) and the maturation of cells to take on specialized functions (differentiation). Health Conditions Related to Genetic Changes DICER1 syndrome Mutations in the DICER1 gene cause DICER1 syndrome. People with this condition have an increased risk of developing many types of tumors, particularly certain tumors of the lungs (pleuropulmonary blastoma); kidneys (cystic nephroma); ovaries (Sertoli- Leydig tumors); and thyroid, a butterfly-shaped gland in the lower neck (multinodular goiter). Most of these mutations lead to an abnormally short Dicer protein that is likely unable to produce miRNA. Without regulation by -
In Vitrocytotoxicity of Ranpirnase (Onconase)
Postepy Hig Med Dosw (online), 2013; 67: 1166-1172 www.phmd.pl e-ISSN 1732-2693 Original Article Received: 2013.06.09 Accepted: 2013.10.13 In vitro cytotoxicity of ranpirnase (onconase) Published: 2013.12.02 in combination with components of R-CHOP regimen against diffuse large B cell lymphoma (DLBCL) cell line* Działanie cytotoksyczne onkonazy w skojarzeniu z lekami schematu R-CHOP na komórki linii chłoniaka rozlanego z dużych komórek B (DLBCL)* Agata Majchrzak1, , , , , , Magdalena Witkowska1,2, , , , Aleksandra Authors’ Contribution: Mędra1, , , Małgorzata Zwolińska1,2, , , , Jakub Bogusz1, , Barbara Study Design Cebula-Obrzut1, , , , , Zbigniew Darzynkiewicz3, , , Tadeusz Robak2, , , , Data Collection 1, , , , , , Statistical Analysis Piotr Smolewski Data Interpretation Manuscript Preparation 1 Department of Experimental Hematology and Literature Search 2 Department of Hematology, Medical University of Lodz, Poland, Funds Collection 3 The Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA Summary Ranpirnase (onconase; ONC) is an endoribonuclease obtained from the frog Rana pipiens. This enzyme exhibits anticancer properties mediated by degradation of cellular RNA and induction of apoptosis. In this study we assessed cytotoxicity of ONC in combination with currently used anticancer drugs on a human diffuse large B-cell lymphoma (DLBCL)-derived cell line (Toledo). Cytotoxic activity was measured by the exclusion of propidium iodide assay while apoptosis was assessed using the annexin-V binding method. Additionally, flow cytometry was used to assess the decline of mitochondrial potential and to determine activation of caspases 3, 8 and 9. It was observed that in vitro treatment with ONC in combination with rituximab, mafosfa- mide, vincristine, doxorubicin, and dexamethasone (drugs corresponding with elements of R-CHOP regimen) resulted in increased cytotoxicity. -
Lp16-PSP, a Member of Yjgf/Yer057c/UK114 Protein
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 September 2017 doi:10.20944/preprints201709.0119.v1 Peer-reviewed version available at Int. J. Mol. Sci. 2017, 18, 2407; doi:10.3390/ijms18112407 1 Article 2 Lp16-PSP, a member of YjgF/YER057c/UK114 Protein 3 Family Induces Apoptosis and p21WAF1/CIP1 mediated G1 4 Cell Cycle Arrest in Human Acute Promyelocytic 5 Leukemia (APL) HL-60 Cells 6 Thomson Patrick Joseph1†, Warren Chanda1†, Abdullah Faqeer Muhammad2, Sadia Kanwal3, Samana 7 Batool1, Meishan Zhang1, MinTao Zhong1 and Min Huang1* 8 1Department of Microbiology, College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning 116044, 9 China 10 2Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning 116044, China 11 3Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning, China 12 †Contributed Equally 13 *Corresponding Author: [email protected]; +8641186110304 14 Abstract: 15 Lp16-PSP from Lentinula edodes strain C91-3 has been reported previously in our laboratory to have selective 16 cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to 17 characterize the Lp16-PSP-induced cell death using HL-60 as model cancer. The results of phase contrast 18 microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high 19 doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the 20 activation of caspase-8, -9 and -3 cascade highlighting the involvement of Fas/FasL-related pathway. 21 Whereas, western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of 22 cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic 23 pathway might be involved in Lp16-PSP-induced apoptosis either. -
Specificity of Escherichia Coli Endoribonuclease Rnase E: in Vivo and in Vitro Analysis of Mutants in a Bacteriophage T4 Mrna Processing Site
Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site Claude P. Ehretsmann/ Agamemnon J. Carpousis,^ and Henry M. Krisch^ Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland Endoribonuclease RNase £ has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A Series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W ^ A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure. [Key Words: Bacteriophage T4 gene 32; mRNA degradation; RNA processing; ribonuclease E] Received August 29, 1991; revised version accepted November 14, 1991. -
Adeno-Associated Virus Rep Proteins Antagonize Phosphatase PP1 To
Adeno-associated virus Rep proteins antagonize PNAS PLUS phosphatase PP1 to counteract KAP1 repression of the latent viral genome Sarah Smith-Moorea, Stuart J. D. Neila, Cornel Fraefelb, R. Michael Lindena, Mathieu Bollenc, Helen M. Rowed, and Els Henckaertsa,1 aDepartment of Infectious Diseases, School of Immunology and Microbial Sciences, King’s College London, SE1 9RT London, United Kingdom; bInstitute of Virology, University of Zurich, 8006 Zurich, Switzerland; cDepartment of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and dDivision of Infection and Immunity, University College London, WC1E 6BT London, United Kingdom Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved March 7, 2018 (received for review December 15, 2017) Adeno-associated virus (AAV) is a small human Dependovirus of various aspects of basic AAV biology. Given that AAV vectors whose low immunogenicity and capacity for long-term persistence likely mimic the latent phase of the viral life cycle, defining the have led to its widespread use as vector for gene therapy. Despite mechanisms involved in the regulation of AAV latency is of great recent successes in AAV-based gene therapy, further im- particular importance for the future design and safety of im- provements in vector technology may be hindered by an inade- proved vectors. In this study, we sought to gain insight into the quate understanding of various aspects of basic AAV biology. AAV regulation of AAV latency by using a screening approach known is unique in that its replication is largely dependent on a helper as “BioID” (17) to identify interaction partners for the AAV2 virus and cellular factors. -
Insights Into the Structure and Architecture of the CCR4–NOT Complex
REVIEW ARTICLE published: 16 May 2014 doi: 10.3389/fgene.2014.00137 Insights into the structure and architecture of the CCR4–NOT complex Kun Xu 1,2 ,Yuwei Bai 1, Aili Zhang 1,2, Qionglin Zhang 2 and Mark G. Bartlam1,2* 1 State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China 2 College of Life Sciences, Nankai University, Tianjin, China Edited by: The CCR4–NOT complex is a highly conserved, multifunctional machinery with a general Martine Anne Collart, University of role in controlling mRNA metabolism. It has been implicated in a number of different Geneva, Switzerland aspects of mRNA and protein expression, including mRNA degradation, transcription Reviewed by: initiation and elongation, ubiquitination, and protein modification. The core CCR4–NOT Walter Lukiw, Louisiana State University, USA complex is evolutionarily conserved and consists of at least three NOT proteins and two Sebastiaan Winkler, University of catalytic subunits.The L-shapedcomplex is characterized by two functional modules bound Nottingham, UK to the CNOT1/Not1 scaffold protein: the deadenylase or nuclease module containing *Correspondence: two enzymes required for deadenylation, and the NOT module. In this review, we will Mark G. Bartlam, College of Life summarize the currently available information regarding the three-dimensional structure Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China and assembly of the CCR4–NOT complex, in order to provide insight into its roles in mRNA e-mail: [email protected] degradation and other biological processes. Keywords: CCR4–NOT complex, mRNA decay, poly(A), deadenylation, NOT module, protein structure INTRODUCTION 0.9–1.2 MDa in size (Liu et al., 1998). -
Rnase E Activity Is Conferred by a Single Polypeptide: Overexpression
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9006-9010, October 1993 Biochemustry RNase E activity is conferred by a single polypeptide: Overexpression, purification, and properties of the ams/rne/hmpl gene product (endoribonuclease/RNA processing) R. S. CORMACK, J. L. GENEREAUX, AND G. A. MACKIE* Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1 Communicated by Myron K. Brakke, June 29, 1993 (receivedfor review May 10, 1993) ABSTRACT Ribonuclease E, an enzyme that processes encoding a high molecular mass protein hypothesized to be pre-SS rRNA from Its precursor, is now believed to be the involved in nucleoid partitioning or cell wall invagination major endoribonuclease participating in mRNA turnover in during division (16). The revised DNA sequence, coterminal Escherichia coli. The product of the ams/rne/hmpl gene, with the 5' end of the two previous sequences, encodes a which is required for RNase E activity, was overexpressed, protein of 1025 amino acid residues whose predicted relative purified to near homogeneity by electroelution from an molecular mass would be 114,000. This polypeptide displays SDS/polyacrylamide gel, and renatured. The purified poly- anomalous electrophoretic mobility in SDS/polyacrylamide peptide possesses nucleolytic activity in vitro with a specificity gels, however, and migrates with an apparent size of 180 kDa identical to that observed for crude RNase E preparations. In (16). It has not been proven that the polypeptide encoded by addition, both UV crosslinking and RNA-protein blotting the ams/rne/hmpl gene is, in fact, a ribonuclease or is unambiguously showed that the Ams/Rne/Hmpl polypeptide intimately associated with the cleavage ofRNA either in vitro has a high affnity for RNA.