DOCSLIB.ORG

The Intron-Mediated Gene Regulation in Saccharomyces Cerevisiae

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Intron-Mediated Gene Regulation in Saccharomyces Cerevisiae The intron-mediated gene regulation in Saccharomyces cerevisiae A thesis presented for the degree of Doctor of Philosophy by Shih-Ching Eva Chen School of Biotechnology and Biomolecular Sciences, University of New South Wales 2011 Table of contents Abstract i Abbreviations ii Acknowledgements iv Publications v Chapter 1 Introduction 1 1.1 The hidden layer of gene regulation, non-coding RNAs 5 1.1.1 Small ncRNAs 7 1.1.2 Long ncRNAs 8 1.1.3 Complex genomic organization 9 1.2 Nuclear introns 12 1.2.1 Significance of introns 12 1.2.2 Function of introns 13 1.2.3 Introns in Saccharomyces cerevisiae 15 1.3 Splicing and the spliceosome 17 1.3.1 Basal machinery 17 1.3.2 Spliceosome assembly and splicing reactions 18 1.3.3 Catalytic centre of spliceosome 21 1.4 The Sm protein family 23 1.4.1 Sm proteins 23 1.4.2 Lsm proteins 26 1.4.3 Other roles of Sm/Lsm complexes 27 1.4.4 Regulation of expression of LSM genes 30 1.5 Aims 34 Chapter 2 Materials and methods 2.1 General materials and methods 36 2.1.1 Materials 36 2.1.2 Sterilization procedures and preparation of materials 37 2.2 Escherichia coli and Saccharomyces cerevisiae strains, media and growth conditions 38 2.2.1 Escherichia coli strains 38 2.2.2 Escherichia coli media and growth conditions 38 2.2.3 Saccharomyces cerevisiae strains 38 2.2.4 Yeast media and growth conditions 39 2.3 General DNA methods 41 2.3.1 General methods and reagents 41 2.3.2 DNA isolation from E. coli or S. cerevisiae 42 2.3.3 Polymerase chain reaction 42 2.3.4 Southern blot analysis 47 2.3.5 DNA sequencing 48 2.4 Generation of strains and plasmids 48 2.4.1 E.coli. transformation 48 2.4.2 Yeast transformation 49 2.4.3 Production of mutant constructs and plasmids 49 2.4.4 Construction of yeast strains 51 2.5 RNA based methods 52 2.5.1 Culture harvest conditions for RNA abundance measurement 52 2.5.2 RNA preparation 52 2.5.3 Quantitative real-time PCR (qRT-PCR) 53 2.5.4 Affymetrix gene chip expression microarray analysis 55 2.6 Mating-type based methods 56 2.6.1 Mating efficiency analysis 56 2.6.2 Measurement of pheromone production 56 2.6.3 Sensitivity test of opposite pheromone 57 2.7 Cell growth assay 57 Chapter 3 Regulation of LSM genes and the function of LSM7 intron on the expression of LSM genes 3.1 Introduction and aims 58 3.2 Production and verification of LSM7 mutant strains 61 3.3 Expression level of LSM genes changes in response to different carbon sources 65 3.4 The requirement for LSM7 coding sequence and intron in maintaining the expression levels of the LSM genes in response to a poor carbon source 67 3.4.1 LSM7 is required for normal expression of LSM genes in response to growth on acetate 67 3.4.2 The LSM7 intron is required for normal expression of LSM genes in response to growth on acetate 70 3.4.3 The LSM7 intron alone is involved in fine-tuning the transcription level of LSM genes in response to growth on acetate 73 3.4.4 Expression of the LSM7 intron improves the growth rate of cells lacking Lsm7 protein 78 3.5 Does the LSM7 intron act as an independent trans-regulator in LSM gene expression ? 80 3.5.1 Expression of LSM7 intron from ADE1 locus alters the expression levels of LSM genes 81 3.5.2 Normal LSM gene expression in cells grown on acetate was not restored with the LSM7 intron expressed from the ADE1 locus in lsm7' and 'i mutants. 85 3.6 Discussion 91 Chapter 4 Regulatory elements of the LSM7 intron 4.1 Introduction and aims 94 4.2 Conservation of the LSM7 intron across yeast species 95 4.3 Linker-scanning mutagenesis of the LSM7 intron 97 4.3.1 Essential sequences of the LSM7 intron in regulation the level of mature LSM7 transcript 98 4.3.2 Expression patterns of LSM genes in response to mutations in the intron under different growth conditions 100 4.3.3 Essential sequences of the LSM7 intron in regulation of LSM genes 105 4.4 Secondary structure prediction of the LSM7 intron 109 4.5 Discussion 111 Chapter 5 Global transcriptional profiling in response to deletion of LSM7 intron 5.1 Introduction and aims 115 5.2. Principle component analysis (PCA) of microarray expression data 116 5.3 Differentially expressed genes in response to the LSM7 intron deletion 118 5.3.1 Differentially expressed genes in response to the LSM7 intron deletion under both media conditions 119 5.3.2 Genes involved in mating-type regulation are altered by the LSM7 intron deletion under both media conditions 124 5.4 The LSM7 intron is required for efficient mating in MATD-cells. 130 5.5 Discussion 133 Chapter 6 Summary and perspectives 135 Reference 141 Key to appendices 165 Abstract Sm-like (Lsm) proteins are critically involved in a variety of RNA-processing events, including splicing, post-transcriptional modification and RNA degradation in organisms that range from bacteria and archea to yeast and humans. In Sacchromyces cerevisiae, the proteins existing in at least two heteroheptameric ring complexes: Lsm1-Lsm7, which promotes mRNA degradation via decapping in the cytoplasm; and, Lsm2-Lsm8, which is required for mRNA splicing in facilitating U4/U6 snRNP in the nucleus. Despite extensive understanding of their function, little is known about the mechanisms that regulate expression of the LSM genes. By constructing a set of mutants that lacked the LSM7 gene, or its intron, or which expressed the intron but not the exons, the LSM7 intron was shown to modulate expression of other LSM genes in trans. The intron located at a separate locus in the ADE1 gene was able to affect expression of some LSM genes although the patterns of expression in cells grown on different carbon sources were not the same as those in the wild type. Sequences within the intron that regulate LSM genes were identified through sequential targeted mutagenesis. The data indicated that the splicing elements are required for full function of the intron. Moreover, a 24 nt region of the intron was found important in controlling the level of expression of the mature LSM7 transcript and other LSM genes in the response to growth on different carbon sources. Microarray analysis was also employed to determine the full extent of the regulatory effect of the intron. Deletion of just the LSM7 intron affected a large group of genes involved in mating in haploids, and a mating efficiency assay provided strong evidence of the requirement of LSM7 intron in mating regulation. However, this regulation was not exerted through control in pheromone production or sensitivity towards opposite mating-type pheromone. These data constitute a step towards understanding not only the regulation of LSM genes but also provide an example of a novel mechanism for gene regulation driven by an intron that can act in trans in a relatively simple eukaryote that lacks the machinery for gene silencing. i Abbreviations 5’SS 5’-splice site 3’SS 3’-splice site aa amino acid BPS branch point sequence bp base pairs d day h hour kb kilo base pair LB Luria-Bertoni broth like-Sm Lsm min minute miRNA micro RNA mRNA messenger RNA ncRNA non-coding RNA nt nucleotides OD600 optical density measured at 600 nm wavelength ORF open reading frame PCR polymerase chain reaction pre-mRNA precursor mRNA qRT-PCR quantitative real-time polymerase chain reaction rRNA ribosomal RNA RNAi RNA-mediated gene silencing s second snRNP small nuclear ribonucleoprotein snRNA small nuclear RNA snoRNA small nucleolar RNA SNP single nucleotide polymorphism SC synthetic complete (minimal) medium SD synthetic define (minimal) medium S. cerevisiae Saccharomyces cerevisiae tRNA transfer RNA UTR untranslated region WT wild-type ii YEPD rich glucose medium YEPA rich acetate medium Gene names for dominant alleles are designated by the standard italicized capitalized three-letter mnemonic, followed by a number (e.g. LSM7). Recessive mutant alleles are denoted in italicized lower case (e.g. lsm7). Deletion is indicated by '. The protein product of the gene is designated by Roman type, with the first letter capitalized (e.g. Lsm7). Open reading frames are designated with three letters, followed by a three digit numerical code and ending in either “c” or “w”. The nomenclature indicates the chromosomal position of the open reading frame (e.g.YNL147W). iii Acknowledgments In has been a privilege to conduct my PhD study in this laboratory. First of all, I would like to pass my greatest thanks to Prof. Ian Dawes for not only the opportunity to work in his lab but also his support, guidance, advice and understanding throughout the years of my study. Without his positive outlook and encouragement, this project would not have been possible. I will always remember your dry ice trick that always work and the moves on the dance floor. My appreciation also goes to all the support and guidance received from Geoff Kornfeld, including small things like making me an RNA special esky, to more importantly supporting me in the project in many ways. Also, I would like to pass my special thanks to Dr. Joyce Chu and Dr. Ruby Lin who have not only kindly provided me their knowledge and experience for most of the RNA works, but also mentally encouraged me throughout my study.
Load more

Recommended publications
  • Analysis of Gene Expression Data for Gene Ontology
    ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Robert Daniel Macholan May 2011 ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION Robert Daniel Macholan Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Zhong-Hui Duan Dr. Chien-Chung Chan _______________________________ _______________________________ Committee Member Dean of the College Dr. Chien-Chung Chan Dr. Chand K. Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Yingcai Xiao Dr. George R. Newkome _______________________________ Date ii ABSTRACT A tremendous increase in genomic data has encouraged biologists to turn to bioinformatics in order to assist in its interpretation and processing. One of the present challenges that need to be overcome in order to understand this data more completely is the development of a reliable method to accurately predict the function of a protein from its genomic information. This study focuses on developing an effective algorithm for protein function prediction. The algorithm is based on proteins that have similar expression patterns. The similarity of the expression data is determined using a novel measure, the slope matrix. The slope matrix introduces a normalized method for the comparison of expression levels throughout a proteome. The algorithm is tested using real microarray gene expression data. Their functions are characterized using gene ontology annotations. The results of the case study indicate the protein function prediction algorithm developed is comparable to the prediction algorithms that are based on the annotations of homologous proteins.
    [Show full text]
  • Allele-Specific Expression of Ribosomal Protein Genes in Interspecific Hybrid Catfish
    Allele-specific Expression of Ribosomal Protein Genes in Interspecific Hybrid Catfish by Ailu Chen A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 1, 2015 Keywords: catfish, interspecific hybrids, allele-specific expression, ribosomal protein Copyright 2015 by Ailu Chen Approved by Zhanjiang Liu, Chair, Professor, School of Fisheries, Aquaculture and Aquatic Sciences Nannan Liu, Professor, Entomology and Plant Pathology Eric Peatman, Associate Professor, School of Fisheries, Aquaculture and Aquatic Sciences Aaron M. Rashotte, Associate Professor, Biological Sciences Abstract Interspecific hybridization results in a vast reservoir of allelic variations, which may potentially contribute to phenotypical enhancement in the hybrids. Whether the allelic variations are related to the downstream phenotypic differences of interspecific hybrid is still an open question. The recently developed genome-wide allele-specific approaches that harness high- throughput sequencing technology allow direct quantification of allelic variations and gene expression patterns. In this work, I investigated allele-specific expression (ASE) pattern using RNA-Seq datasets generated from interspecific catfish hybrids. The objective of the study is to determine the ASE genes and pathways in which they are involved. Specifically, my study investigated ASE-SNPs, ASE-genes, parent-of-origins of ASE allele and how ASE would possibly contribute to heterosis. My data showed that ASE was operating in the interspecific catfish system. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5,420 (8.2%) and 13,390 (7.5%) SNPs were identified as significant ASE-SNPs, respectively.
    [Show full text]
  • Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-Like Mouse Models: Tracking the Role of the Hairless Gene
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene Yutao Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Liu, Yutao, "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino- like Mouse Models: Tracking the Role of the Hairless Gene. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1824 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yutao Liu entitled "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Brynn H. Voy, Major Professor We have read this dissertation and recommend its acceptance: Naima Moustaid-Moussa, Yisong Wang, Rogert Hettich Accepted for the Council: Carolyn R.
    [Show full text]
  • Molecular Basis for the Distinct Cellular Functions of the Lsm1-7 and Lsm2-8 Complexes
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.055376; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes Eric J. Montemayor1,2, Johanna M. Virta1, Samuel M. Hayes1, Yuichiro Nomura1, David A. Brow2, Samuel E. Butcher1 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. 2Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. Correspondence should be addressed to E.J.M. ([email protected]) and S.E.B. ([email protected]). Abstract Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines.
    [Show full text]
  • TACC1–Chtog–Aurora a Protein Complex in Breast Cancer
    Oncogene (2003) 22, 8102–8116 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc TACC1–chTOG–Aurora A protein complex in breast cancer Nathalie Conte1,Be´ ne´ dicte Delaval1, Christophe Ginestier1, Alexia Ferrand1, Daniel Isnardon2, Christian Larroque3, Claude Prigent4, Bertrand Se´ raphin5, Jocelyne Jacquemier1 and Daniel Birnbaum*,1 1Department of Molecular Oncology, U119 Inserm, Institut Paoli-Calmettes, IFR57, Marseille, France; 2Imaging Core Facility, Institut Paoli-Calmettes, Marseille, France; 3E229 Inserm, CRLC Val d’Aurelle/Paul Lamarque, Montpellier, France; 4Laboratoire du cycle cellulaire, UMR 6061 CNRS, IFR 97, Faculte´ de Me´decine, Rennes, France; 5Centre de Ge´ne´tique Mole´culaire, Gif-sur-Yvette, France The three human TACC (transforming acidic coiled-coil) metabolism, including mitosis and intracellular trans- genes encode a family of proteins with poorly defined port of molecules, is progressing but many components functions that are suspected to play a role in oncogenesis. remain to be discovered and characterized. We describe A Xenopus TACC homolog called Maskin is involved in here the interaction of the TACC1 protein with several translational control, while Drosophila D-TACC interacts protein partners that makes it a good candidate to with the microtubule-associated protein MSPS (Mini participate in microtubule-associated processes in nor- SPindleS) to ensure proper dynamics of spindle pole mal and tumoral cells. microtubules during cell division. We have delineated here In
    [Show full text]
  • Deep Multiomics Profiling of Brain Tumors Identifies Signaling Networks
    ARTICLE https://doi.org/10.1038/s41467-019-11661-4 OPEN Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes Hong Wang 1,2,3, Alexander K. Diaz3,4, Timothy I. Shaw2,5, Yuxin Li1,2,4, Mingming Niu1,4, Ji-Hoon Cho2, Barbara S. Paugh4, Yang Zhang6, Jeffrey Sifford1,4, Bing Bai1,4,10, Zhiping Wu1,4, Haiyan Tan2, Suiping Zhou2, Laura D. Hover4, Heather S. Tillman 7, Abbas Shirinifard8, Suresh Thiagarajan9, Andras Sablauer 8, Vishwajeeth Pagala2, Anthony A. High2, Xusheng Wang 2, Chunliang Li 6, Suzanne J. Baker4 & Junmin Peng 1,2,4 1234567890():,; High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT down- stream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics inte- grative approaches, and providing novel tumor vulnerabilities.
    [Show full text]
  • Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases A, D, and E in Saccharomyces Cerevisiae
    INVESTIGATION Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases a, d, and e in Saccharomyces cerevisiae Marion Dubarry,* Conor Lawless,* A. Peter Banks,† Simon Cockell,‡ and David Lydall*,1 *Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, †High Throughput Screening Facility, Newcastle Biomedicine, and ‡Bioinformatics Support Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom ORCID IDs: 0000-0002-4186-8506 (C.L.); 0000-0003-2478-085X (D.L.) ABSTRACT Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase KEYWORDS a-primase (Pol a) and DNA polymerase d (Pol d) replicate the lagging-strand and Pol a and DNA polymerase e DNA replication (Pol e) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed DNA polymerase genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We Profilyzer combined temperature-sensitive mutations affecting the three replicative polymerases, Pol a,Pold,and DIXY Pol e with genome-wide collections of null and reduced function mutations. We identify large numbers of quantitative genetic interactions that inform about the roles that specific genes play to help Pol a,Pold, and Pol e function. fitness analyses Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not (QFA) represent the majority of the genetic interactions identified. Instead our data support a model for division of Saccharomyces labor between the different DNA polymerases during DNA replication. For example, our genetic interaction cerevisiae data are consistent with biochemical data showing that Pol e is more important to the Pre-Loading complex than either Pol a or Pol d.
    [Show full text]
  • TACC-Ling Microtubule Dynamics and Centrosome Function
    Review The TACC proteins: TACC-ling microtubule dynamics and centrosome function Isabel Peset1 and Isabelle Vernos1,2 1 Cell and Developmental Biology Program, Centre for Genomic Regulation (CRG), University Pompeu Fabra (UPF), Dr Aiguader 88, Barcelona 08003, Spain 2 Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Passeig Lluis Companys 23, 08010 Barcelona, Spain A major quest in cell biology is to understand the Transforming acidic coiled-coil (TACC) proteins emerged molecular mechanisms underlying the high plasticity initially as a group of proteins implicated in cancer. The of the microtubule network at different stages of the first member of the TACC family to be discovered was cell cycle, and during and after differentiation. Initial identified in a search of genomic regions that are amplified reports described the centrosomal localization of in breast cancer. It was named transforming acidic coiled- proteins possessing transforming acidic coiled-coil coil 1 (TACC1) because of its highly acidic nature, the (TACC) domains. This discovery prompted several presence of a predicted coiled-coil domain at its C terminus groups to examine the role of TACC proteins during cell (now known as the TACC domain), and its ability to division, leading to indications that they are important promote cellular transformation [6]. TACC proteins are players in this complex process in different organisms. present in different organisms, ranging from yeasts to Here, we review the current understanding of the role of mammals. There is only one TACC protein in the nema- TACC proteins in the regulation of microtubule tode Caenorhabditis elegans (TAC-1), in Drosophila mel- dynamics, and we highlight the complexity of centro- anogaster (D-TACC), in Xenopus laevis (Maskin), and some function.
    [Show full text]
  • A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family
    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase.
    [Show full text]
  • Molecular Basis for the Distinct Cellular Functions of the Lsm1-7 and Lsm2-8 Complexes
    Downloaded from rnajournal.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes Eric J. Montemayor1,2, Johanna M. Virta1, Samuel M. Hayes1, Yuichiro Nomura1, David A. Brow2, Samuel E. Butcher1 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. 2Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. Correspondence should be addressed to E.J.M. ([email protected]) and S.E.B. ([email protected]). Abstract Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3′ end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites.
    [Show full text]
  • New Insights on Human Essential Genes Based on Integrated Multi
    bioRxiv preprint doi: https://doi.org/10.1101/260224; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. New insights on human essential genes based on integrated multi- omics analysis Hebing Chen1,2, Zhuo Zhang1,2, Shuai Jiang 1,2, Ruijiang Li1, Wanying Li1, Hao Li1,* and Xiaochen Bo1,* 1Beijing Institute of Radiation Medicine, Beijing 100850, China. 2 Co-first author *Correspondence: [email protected]; [email protected] Abstract Essential genes are those whose functions govern critical processes that sustain life in the organism. Comprehensive understanding of human essential genes could enable breakthroughs in biology and medicine. Recently, there has been a rapid proliferation of technologies for identifying and investigating the functions of human essential genes. Here, according to gene essentiality, we present a global analysis for comprehensively and systematically elucidating the genetic and regulatory characteristics of human essential genes. We explain why these genes are essential from the genomic, epigenomic, and proteomic perspectives, and we discuss their evolutionary and embryonic developmental properties. Importantly, we find that essential human genes can be used as markers to guide cancer treatment. We have developed an interactive web server, the Human Essential Genes Interactive Analysis Platform (HEGIAP) (http://sysomics.com/HEGIAP/), which integrates abundant analytical tools to give a global, multidimensional interpretation of gene essentiality. bioRxiv preprint doi: https://doi.org/10.1101/260224; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • Dramatically Reduced Spliceosome in Cyanidioschyzon Merolae
    Dramatically reduced spliceosome in PNAS PLUS Cyanidioschyzon merolae Martha R. Starka, Elizabeth A. Dunnb, William S. C. Dunna, Cameron J. Grisdalec, Anthony R. Danielea, Matthew R. G. Halsteada, Naomi M. Fastc, and Stephen D. Radera,b,1 aDepartment of Chemistry, University of Northern British Columbia, Prince George, BC, V2N 4Z9 Canada; and Departments of bBiochemistry and Molecular Biology, and cBotany, University of British Columbia, Vancouver, BC, V6T 1Z4 Canada Edited by Joan A. Steitz, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved February 9, 2015 (received for review September 1, 2014) The human spliceosome is a large ribonucleoprotein complex that C. merolae is an acidophilic, unicellular red alga that grows at catalyzes pre-mRNA splicing. It consists of five snRNAs and more temperatures of up to 56 °C (6). At 16.5 million base pairs, its than 200 proteins. Because of this complexity, much work has genome is similar in size to that of S. cerevisiae and contains focusedontheSaccharomyces cerevisiae spliceosome, viewed as a comparable number of genes; however only one tenth as many a highly simplified system with fewer than half as many splicing introns were annotated in C. merolae: 26 intron-containing factors as humans. Nevertheless, it has been difficult to ascribe genes, 0.5% of the genome (6). The small number of introns in a mechanistic function to individual splicing factors or even to dis- C. merolae raises the questions of whether the full complexity cern which are critical for catalyzing the splicing reaction. We have of the canonical splicing machinery has been maintained or C merolae identified and characterized the splicing machinery from the red alga whether .
    [Show full text]