Mechanisms of Mrna Polyadenylation
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Mutation of the AAUAAA Polyadenylation Signal Depresses in Vitro Splicing of Proximal but Not Distal Introns
Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns Maho Niwa and Susan M. Berget Marrs McClean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 USA To investigate the relationship between splicing and polyadenylation during the production of vertebrate mRNAs, we examined the effect of mutation of a poly(A) site on splicing of upstream introns. Mutation of the AAUAAA polyadenylation consensus sequence inhibited in vitro splicing of an upstream intron. The magnitude of the depression depended on the magnesium concentration. Dependence of splicing on polyadenylation signals suggests the existence of interaction between polyadenylation and splicing factors. In multi-intron precursor RNAs containing duplicated splice sites, mutation of the poly(A) site inhibited removal of the last intron, but not the removal of introns farther upstream. Inhibition of removal of only the last intron suggests segmental recognition of multi-exon precursor RNAs and is consistent with previous suggestions that signals at both ends of an exon are required for effective splicing of an upstream intron. [Key Words: Polyadenylation signal; splicing; vertebrate RNAs] Received July 31, 1991; revised version accepted September 10, 1991. The majority of vertebrate mRNAs undergo both splic- Using chimeric RNAs competent for both in vitro ing and polyadenylation during maturation. The rela- splicing and polyadenylation, we recently reported that tionship between the two processing steps has been un- in vitro polyadenylation is stimulated when an intron is clear. Polyadenylation has been reported to occur shortly placed upstream of a poly(A) site (Niwa et al. -
Mayr, Annu Rev Genet 2017
GE51CH09-Mayr ARI 12 October 2017 10:21 Annual Review of Genetics Regulation by 3-Untranslated Regions Christine Mayr Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; email: [email protected] Annu. Rev. Genet. 2017. 51:171–94 Keywords First published as a Review in Advance on August noncoding RNA, regulatory RNA, alternative 3-UTRs, alternative 30, 2017 polyadenylation, cotranslational protein complex formation, cellular The Annual Review of Genetics is online at organization, mRNA localization, RNA-binding proteins, cooperativity, genet.annualreviews.org accessibility of regulatory elements https://doi.org/10.1146/annurev-genet-120116- 024704 Abstract Copyright c 2017 by Annual Reviews. 3 -untranslated regions (3 -UTRs) are the noncoding parts of mRNAs. Com- All rights reserved pared to yeast, in humans, median 3-UTR length has expanded approx- imately tenfold alongside an increased generation of alternative 3-UTR isoforms. In contrast, the number of coding genes, as well as coding region length, has remained similar. This suggests an important role for 3-UTRs in the biology of higher organisms. 3-UTRs are best known to regulate ANNUAL REVIEWS Further diverse fates of mRNAs, including degradation, translation, and localiza- Click here to view this article's tion, but they can also function like long noncoding or small RNAs, as has Annu. Rev. Genet. 2017.51:171-194. Downloaded from www.annualreviews.org online features: • Download figures as PPT slides been shown for whole 3 -UTRs as well as for cleaved fragments. Further- • Navigate linked references • Download citations more, 3 -UTRs determine the fate of proteins through the regulation of Access provided by Memorial Sloan-Kettering Cancer Center on 11/30/17. -
Polymerse Activity
University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2005 CHARACTERIZATION OF PLANT POLYADENYLATION TRANSACTING FACTORS-FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY Kevin Patrick Forbes University of Kentucky Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Forbes, Kevin Patrick, "CHARACTERIZATION OF PLANT POLYADENYLATION TRANSACTING FACTORS- FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY" (2005). University of Kentucky Doctoral Dissertations. 444. https://uknowledge.uky.edu/gradschool_diss/444 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Kevin Patrick Forbes The Graduate School University of Kentucky 2004 CHARACTERIZATION OF PLANT POLYADENYLATION TRANS- ACTING FACTORS-FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY _________________________________________ ABSTRACT OF DISSERTATION _________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Kevin Patrick Forbes Lexington, Kentucky Director: Dr. Arthur G. Hunt, Professor of Agronomy Lexington, Kentucky 2004 Copyright ” Kevin Patrick Forbes 2004 ABSTRACT OF DISSERTATION CHARACTERIZATION OF PLANT POLYADENYLATION TRANS-ACTING FACTORS-FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. -
Regulation of Translation by the 3' Untranslated Region of Barley Yellow Dwarf Virus-PAV RNA Shanping Wang Iowa State University
Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1996 Regulation of translation by the 3' untranslated region of barley yellow dwarf virus-PAV RNA Shanping Wang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Molecular Biology Commons, and the Plant Pathology Commons Recommended Citation Wang, Shanping, "Regulation of translation by the 3' untranslated region of barley yellow dwarf virus-PAV RNA " (1996). Retrospective Theses and Dissertations. 11426. https://lib.dr.iastate.edu/rtd/11426 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afTect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. -
Symplekin and Transforming Acidic Coiled-Coil Containing Protein 3 Support the Cancer Cell Mitotic Spindle
SYMPLEKIN AND TRANSFORMING ACIDIC COILED-COIL CONTAINING PROTEIN 3 SUPPORT THE CANCER CELL MITOTIC SPINDLE Kathryn M. Cappell A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Pharmacology, School of Medicine. Chapel Hill 2011 Approved by: Advisor: Dr. Angelique Whitehurst Reader: Dr. David Siderovski Reader: Dr. Channing Der Reader: Dr. Pilar Blancafort Reader: Dr. Mohanish Deshmukh ABSTRACT KATHRYN CAPPELL: Symplekin and Transforming Acidic Coiled-Coil Containing Protein 3 Support the Cancer Cell Mitotic Spindle (Under the direction of Dr. Angelique Whitehurst) An increased rate of proliferation in cancer cells, combined with abnormalities in spindle architecture, places tumors under increased mitotic stress. Previously, our laboratory performed a genome-wide paclitaxel chemosensitizer screen to identify genes whose depletion sensitizes non- small cell lung cancer (NSCLC) cells to mitotic stress induced by paclitaxel treatment. This screen uncovered a cohort of genes that are required for viability only in the presence of paclitaxel. Two genes uncovered in this screen were the polyadenylation scaffold symplekin and the gametogenic protein transforming acidic coiled-coil containing protein 3 (TACC3). Herein, we examine the impact of polyadenylation and gametogenesis on the tumor cell mitotic spindle. First, we demonstrate that depletion of SYMPK and other polyadenylation components sensitizes many NSCLC cells, but not normal immortalized lines, to paclitaxel by inducing mitotic errors and leading to abnormal mitotic progression. Second, we demonstrate that multiple gametogenic genes are required for normal microtubule dynamics and mitotic spindle formation in the presence of paclitaxel. -
Anti-CSTF2 / Cstf 64 Antibody (ARG58549)
Product datasheet [email protected] ARG58549 Package: 50 μl anti-CSTF2 / CstF 64 antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes CSTF2 / CstF 64 Tested Reactivity Hu Predict Reactivity Ms, Rat, Cow, Dog, Gpig, Hrs, Rb Tested Application IHC-P, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name CSTF2 / CstF 64 Antigen Species Human Immunogen Synthetic peptide around the N-terminal region of Human CSTF2 / CstF 64. (within the following sequence: VDPEIALKILHRQTNIPTLIAGNPQPVHGAGPGSGSNVSMNQQNPQAPQA) Conjugation Un-conjugated Alternate Names CstF-64; CF-1 64 kDa subunit; Cleavage stimulation factor 64 kDa subunit; CSTF 64 kDa subunit; Cleavage stimulation factor subunit 2 Application Instructions Predict Reactivity Note Predicted homology based on immunogen sequence: Cow: 85%; Dog: 85%; Guinea Pig: 86%; Horse: 86%; Mouse: 86%; Rabbit: 100%; Rat: 93% Application table Application Dilution IHC-P 4 - 8 µg/ml WB 0.2 - 1 µg/ml Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control 293T Calculated Mw 61 kDa Properties Form Liquid Purification Affinity purified. Buffer PBS, 0.09% (w/v) Sodium azide and 2% Sucrose. Preservative 0.09% (w/v) Sodium azide Stabilizer 2% Sucrose www.arigobio.com 1/3 Concentration Batch dependent: 0.5 - 1 mg/ml Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C or below. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. -
The Nuclear Poly(A) Binding Protein of Mammals, but Not of Fission Yeast, Participates in Mrna Polyadenylation
Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT The nuclear poly(A) binding protein of mammals, but not of fission yeast, participates in mRNA polyadenylation UWE KÜHN, JULIANE BUSCHMANN, and ELMAR WAHLE Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany ABSTRACT The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of ∼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors. Keywords: poly(A) binding protein; poly(A) polymerase; mRNA polyadenylation; pre-mRNA 3′; processing INTRODUCTION by poly(A) polymerase with the help of the cleavage and poly- adenylation specificity factor (CPSF), which binds the polya- The poly(A) tails of eukaryotic mRNAs are covered by specif- denylation signal AAUAAA. -
Quantitative SUMO Proteomics Reveals the Modulation of Several
www.nature.com/scientificreports OPEN Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated Received: 10 October 2017 Accepted: 28 March 2018 proteins and an anti-senescence Published: xx xx xxxx function of UBC9 Francis P. McManus1, Véronique Bourdeau2, Mariana Acevedo2, Stéphane Lopes-Paciencia2, Lian Mignacca2, Frédéric Lamoliatte1,3, John W. Rojas Pino2, Gerardo Ferbeyre2 & Pierre Thibault1,3 Several regulators of SUMOylation have been previously linked to senescence but most targets of this modifcation in senescent cells remain unidentifed. Using a two-step purifcation of a modifed SUMO3, we profled the SUMO proteome of senescent cells in a site-specifc manner. We identifed 25 SUMO sites on 23 proteins that were signifcantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation. Many cellular mechanisms of defense have evolved to reduce the onset of tumors and potential cancer develop- ment. One such mechanism is cellular senescence where cells undergo cell cycle arrest in response to various stressors1,2. Multiple triggers for the onset of senescence have been documented. While replicative senescence is primarily caused in response to telomere shortening3,4, senescence can also be triggered early by a number of exogenous factors including DNA damage, elevated levels of reactive oxygen species (ROS), high cytokine signa- ling, and constitutively-active oncogenes (such as H-RAS-G12V)5,6. -
Related Regulators of 3′ UTR Processing with KAPAC Andreas J
Gruber et al. Genome Biology (2018) 19:44 https://doi.org/10.1186/s13059-018-1415-3 METHOD Open Access Discovery of physiological and cancer- related regulators of 3′ UTR processing with KAPAC Andreas J. Gruber† , Ralf Schmidt†, Souvik Ghosh, Georges Martin, Andreas R. Gruber, Erik van Nimwegen and Mihaela Zavolan* Abstract 3′ Untranslated regions (3' UTRs) length is regulated in relation to cellular state. To uncover key regulators of poly(A) site use in specific conditions, we have developed PAQR, a method for quantifying poly(A) site use from RNA sequencing data and KAPAC, an approach that infers activities of oligomeric sequence motifs on poly(A) site choice. Application of PAQR and KAPAC to RNA sequencing data from normal and tumor tissue samples uncovers motifs that can explain changes in cleavage and polyadenylation in specific cancers. In particular, our analysis points to polypyrimidine tract binding protein 1 as a regulator of poly(A) site choice in glioblastoma. Keywords: Cleavage and polyadenylation, APA, CFIm, KAPAC, PAQR, HNRNPC, PTBP1, Prostate adenocarcinoma, Glioblastoma, Colon adenocarcinoma Background signal, consisting of the CPSF1, CPSF4, FIP1L1, and The 3′ ends of most eukaryotic mRNAs are generated WDR33 proteins, has been identified [6, 7]. through endonucleolytic cleavage and polyadenylation Most genes have multiple poly(A) sites (PAS), which (CPA) [1–3]. These steps are carried out in mammalian are differentially processed across cell types [8], likely cells by a 3′ end processing complex composed of the due to cell type-specific interactions with RNA-binding cleavage and polyadenylation specificity factor (which in- proteins (RBPs). The length of 3′ UTRs is most strongly cludes the proteins CPSF1 (also known as CPSF160), dependent on the mammalian cleavage factor I (CFIm), CPSF2 (CPSF100), CPSF3 (CPSF73), CPSF4 (CPSF30), which promotes the use of distal poly(A) sites [5, 9–12]. -
Development and Validation of an RNA Binding Protein- Associated Prognostic Model for Hepatocellular Carcinoma
Journal of Clinical and Translational Hepatology 2021 DOI: 10.14218/JCTH.2020.00103 Original Article Development and Validation of an RNA Binding Protein- associated Prognostic Model for Hepatocellular Carcinoma Hao Zhang , Peng Xia, Weijie Ma and Yufeng Yuan* Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China Received: 7 November 2020 | Revised: 24 February 2021 | Accepted: 26 March 2021 | Published: 13 April 2021 Abstract ated prognostic model for hepatocellular carcinoma. J Clin Transl Hepatol 2021. doi: 10.14218/JCTH.2020.00103. Background and Aims: The survival rate of patients with hepatocellular carcinoma is variable. The abnormal expres- sion of RNA-binding proteins (RBPs) is closely related to the occurrence and development of malignant tumors. The pri- Introduction mary aim of this study was to identify RBPs related to the prognosis of liver cancer and to construct a prognostic model Worldwide, liver cancer is the fourth leading cause of can- of liver cancer. Methods: We downloaded the hepatocellular cer-related deaths and has the sixth highest incidence.1 carcinoma gene sequencing data from The Cancer Genome It is estimated that 840,000 new cases of liver cancer are Atlas (cancergenome.nih.gov/) database, constructed a diagnosed and at least 780,000 people die of liver cancer protein-protein interaction network, and used Cytoscape to every year, with China accounting for 47% of the total num- realize the visualization. From among 325 abnormally ex- ber of liver cancer cases as well as the related mortality.2,3 pressed genes for RBPs, 9 (XPO5, enhancer of zeste 2 poly- Hepatocellular carcinoma (HCC) is the predominant type of comb repressive complex 2 subunit [EZH2], CSTF2, BRCA1, primary liver cancer, accounting for approximately 90% of RRP12, MRPL54, EIF2AK4, PPARGC1A, and SEPSECS) were all liver cancer cases.4 Although great progress has been selected for construction of the prognostic model. -
Antisense Oligonucleotide-Based Therapeutic Against Menin for Triple-Negative Breast Cancer Treatment
biomedicines Article Antisense Oligonucleotide-Based Therapeutic against Menin for Triple-Negative Breast Cancer Treatment Dang Tan Nguyen 1,†, Thi Khanh Le 1,2,† , Clément Paris 1,†, Chaïma Cherif 1 , Stéphane Audebert 3 , Sandra Oluchi Udu-Ituma 1,Sébastien Benizri 4 , Philippe Barthélémy 4 , François Bertucci 1, David Taïeb 1,5 and Palma Rocchi 1,* 1 Predictive Oncology Laboratory, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm UMR 1068, CNRS UMR 7258, Institut Paoli-Calmettes, Aix-Marseille University, 27 Bd. Leï Roure, 13273 Marseille, France; [email protected] (D.T.N.); [email protected] (T.K.L.); [email protected] (C.P.); [email protected] (C.C.); [email protected] (S.O.U.-I.); [email protected] (F.B.); [email protected] (D.T.) 2 Department of Life Science, University of Science and Technology of Hanoi (USTH), Hanoi 000084, Vietnam 3 Marseille Protéomique, Centre de Recherche en Cancérologie de Marseille, INSERM, CNRS, Institut Paoli-Calmettes, Aix-Marseille University, 13009 Marseille, France; [email protected] 4 ARNA Laboratory, INSERM U1212, CNRS UMR 5320, University of Bordeaux, 33076 Bordeaux, France; [email protected] (S.B.); [email protected] (P.B.) 5 Biophysics and Nuclear Medicine Department, La Timone University Hospital, European Center for Research in Medical Imaging, Aix-Marseille University, 13005 Marseille, France * Correspondence: [email protected]; Tel.: +33-626-941-287 † These authors contributed equally. Citation: Nguyen, D.T.; Le, T.K.; Paris, C.; Cherif, C.; Audebert, S.; Abstract: The tumor suppressor menin has dual functions, acting either as a tumor suppressor or Oluchi Udu-Ituma, S.; Benizri, S.; as an oncogene/oncoprotein, depending on the oncological context. -
Translation from the 5′ Untranslated Region Shapes the Integrated Stress Response Shelley R
RESEARCH ◥ duce tracer peptides. These translation prod- RESEARCH ARTICLE SUMMARY ucts are processed and loaded onto major histocompatibility complex class I (MHC I) molecules in the ER and transit to the cell STRESS RESPONSE surface, where they can be detected by specific T cell hybridomas that are activated and quan- ′ tified using a colorimetric reagent. 3T provides Translation from the 5 untranslated an approach to interrogate the thousands of predicted uORFs in mammalian genomes, char- region shapes the integrated acterize the importance of uORF biology for regulation, and generate fundamental insights stress response into uORF mutation-based diseases. Shelley R. Starck,* Jordan C. Tsai, Keling Chen, Michael Shodiya, Lei Wang, RESULTS: 3T proved to be a sensitive and Kinnosuke Yahiro, Manuela Martins-Green, Nilabh Shastri,* Peter Walter* robust indicator of uORF expression. We mea- sured uORF expression in the 5′ UTR of INTRODUCTION: Protein synthesis is con- mRNAs, such as ATF4 and CHOP, that harbor mRNAs at multiple distinct regions, while trolled by a plethora of developmental and small upstream open reading frames (uORFs) ◥ simultaneously detecting environmental conditions. One intracellular in their 5′ untranslated regions (5′ UTRs). Still ON OUR WEB SITE expression of the CDS. We signaling network, the integrated stress re- other mRNAs sustain translation despite ISR Read the full article directly measured uORF sponse (ISR), activates one of four kinases in activation. We developed tracing translation at http://dx.doi. peptide expression from response to a variety of distinct stress stimuli: by T cells (3T) as an exquisitely sensitive tech- org/10.1126/ ATF4 mRNA and showed the endoplasmic reticulum (ER)–resident kinase nique to probe the translational dynamics of science.aad3867 that its translation per- .................................................