DOCSLIB.ORG

Considerations for the Design and Cgmp Manufacturing of Mrna Therapeutics Josh M

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Considerations for the Design and Cgmp Manufacturing of Mrna Therapeutics Josh M Considerations for the Design and cGMP Manufacturing of mRNA Therapeutics Josh M. Whisenand, Krist T. Azizian, Jordana M. Henderson, Sabrina Shore, Dongwon Shin, Alexandre Lebedev, Anton P. McCaffrey and Richard I. Hogrefe TriLink BioTechnologies, San Diego, CA 92121, USA. Abstract Capping Methods mRNA Purification Recently, there has been significant interest in the use of messenger RNA (mRNA) as anex vivo and in vivo therapeutic. Since mRNA is expressed in the cytoplasm it may be particularly useful for improving • Enzymatic capping characteristics Purification is required to remove: salts, NTPs, cap analogs, gene expression in difficult to transfect non-dividing cells. In contrast to plasmid or viral vectors, there is no risk of insertional mutagenesis or subsequent oncogenesis upon mRNA transfection and the - Utilizes Vaccinia virus capping enzyme and 2’ O-methyltransferase proteins, truncated mRNA products, residual DNA and transient nature of mRNA expression is desirable for genome editing (CRISPR/Cas9, ZFNs and TALENs) enzymes and vaccines. In each case, the goal is to produce a synthetic RNA that mimics a natural mRNA. sometimes double stranded RNA - Can potentially achieve 100 % capping with a natural unmodified In anticipation of clinical testing, it is essential to use sequence designs, raw materials and cap structure manufacturing processes that are scalable. Early on, critical decisions must be made about 1) - Very costly at scale with significant batch- to-batch enzymatic • TriLink has developed two complementary transcription template designs, 2) capping methodologies, 3) sequence optimization, 4) chemical modifications to evade innate immune responses, 5) polyadenylation methods and 6) purification - Accessibility of 5’ end important for efficient capping chromatographic mRNA purification methods: methods. Co-transcriptional capping For optimal expression in cells or target organs, transfected mRNAs must avoid detection by pattern - LC-Isolation recognition receptors (PRRs) that evolved to sense improperly capped RNAs and double stranded RNA. - Removes residual proteins, DNA, and NTPs PRR activation leads to cytokine production, translational arrest and cell toxicity or death. Mammalian • ARCA capping characteristics - Highly scalable mRNAs are modified post-transcriptionally to contain modified nucleotides (e.g. pseudouridine and - Capping fairly reproducible (60-80 % capping, 20-40% uncapped) 5-methylcytidine). These modifications can reduce activation of PRRs and allow maximal translation. - There is a significant amount of transcriptional “stuttering” - Reverse phase HPLC (RP-HPLC) During RNA capping, Cap 0 (m7GpppN) is formed as an intermediate. Methylation of the 2’ position of - Cap contains an unnatural 3’ O-methyl group - Decreases double-stranded RNA the first nucleotide forms Cap 1 (found in 100% of transcripts) and Cap 2 (found in 50% of transcripts) - Transcript must start with G of endogenous mRNAs. mRNAs generated with commercially available cap analogs (ARCA) contain - Enriches for full length mRNA Cap 0 structures that can be immunogenic. Recombinant enzymes used to generate Cap 1 mRNA - Longer sequences more challenging to purify are expensive, do not always go to completion and the RNA must be purified prior to capping. A novel • CleanCap™ 3rd generation cap analog co-transcriptional capping method that yields Cap 1 or Cap 2 with high efficiency and lower costs in a - Capping efficiencies typically 90-99% “one pot” reaction will be discussed. - Can produce Cap 0, Cap 1, or Cap 2 First generation mRNAs were modified with pseudouridine or 5-methylcytidine/pseudouridine and had - Yields a natural unmodified cap structure Cap 0 structures. Data from improved second generation Cap 1 mRNAs containing a combination - Allows any base (A, C, G, or U) at 5’ end of sequence engineering and chemical modifications will be presented. We will provide a broad m6 m6 roadmap for the application of these principles to the design and manufacturing of novel mRNA - Allows A or Am at 5’ end Figure 4: RP-HPLC Purification Yields More therapeutics. - More affordable than enzymatic capping Homogenous Luciferase mRNA and Depletes dsRNA O B (Slot Blot) Cap1 HO OH O P O 1 - O O P O O - P O O O - O O OMe Background: Why mRNA Therapeutics? H2N N N O B O P O 2 HN O mRNA is a popular new tool for gene expression because it: N O - Does not have a risk of insertional mutagenesis O Pre HPLC HO OH Post HPLC - Can transfect difficult cells such as non-dividing cells O B - Is transient Cap2 HO OH O P O 1 - O O P O O - P O O • Applications O - O O OMe H2N N N O - Genome editing (Transposons, Cre, ZFNs, TALENs and CRISPR/Cas9) B O P O 2 HN O - Gene replacement N O - Vaccines O O OMe dsRNA slot blot HPLC vs. non-HPLC B wt wt + HPLC PseudoU PseudoU + 5moU + O P O 3 HPLC 5moU HPLC • Limitations O O 4000 ng - Innate immune response to unmodified mRNA HO OH 1000 ng • Solutions 250 ng - Proper capping 8 ng 2 ng 0.5 ng 0 ng dsRNA - Chemical modification of mRNA can prevent innate immune Standard Figure 2: CleanCap™, Enzymatic and ARCA, Slot blot analysis of dsRNA present in mRNA samples. Protocol adapted from stimulation Pub Med ID 23296926. Capping Comparison - Removal of dsRNA CleanCap 6 x 107 Activity of U depleted Renilla Luciferase (THP-1 Dual, 100ng mRNA) Enzymatic non-HPLC ARCA HPLC Chemical Modification and Sequence 4 x 107 Optimization of mRNA Can Prevent Innate Immune Stimulation Raw Light Units 2 x 107 Transfection of cells with unmodified RNAs can lead to cell death due to activation of innate immune pathways 0 wt wt • Toll-like receptors 3, 7 & 8 recognize different RNA forms PsU PsU 5moU 5moU neg ctrl - Found in endosomes where some viruses enter cells LC-MS analysis • Cytosolic sensors - Protein Kinase R (PKR): dsRNA Figure 3: Template Design Cas9 - MDA5: dsRNA Purification Conclusions - IFITs: unmethylated cap structures - RIG-I: 5’ triphosphate • For some applications, LC-Isolation may be of sufficient quality - cGAS/STING – cytosolic DNA especially if using uridine-depleted 5-methoxyuridine-substituted mRNAs. May not require RP-HPLC • Endotoxin free • Kanamycin resistance • For other applications, RP-HPLC purification may be required to • Epitope tags may be undesirable remove double-stranded RNA Figure 1: mRNA Synthesis by in vitro Transcription • Is GMP plasmid required? • RP-HPLC purification reduces interferon induction in THP-1 cells in • Cell banking many cases • Think about plasmid sourcing early • When selecting synthesis scale, account for purification yield 1. T7 RNA polymerase 2. Inorganic pyrophosphatase (optional) 3. RNase inhibitor (optional) Figure 5: Phases GMP mRNA Drug Development 4. Transcription template 5. Standard and/or modified NTPs Program 6. Cap analogs (optional) Chemical Modification in Combination with Sequence Optimization Year 1 Year 2 • TriLink offers over 200 modified NTPs Agreements Function of mRNA Cap Structures • Uridine depletion of template in combination with chemical 3 - 4 Months modification increases activity • mRNA cap structures are involved in modulating Process Development - Nuclear Export - Splicing – RNA Turnover - Translational Regulation Luciferase activity in THP-1 cells 4 - 5 Months 6.0 x 107 Standard FLuc U depleted FLuc Scale-up Manufacturing • Cap 1 and Cap 2 are important for self/non-self 2 - 3 Months 4.0 x 107 recognition by the innate immune system 2.0 x 107 GMP Document - IFITs recognize non-methylated caps Generation 2 - 3 Months - Cap 0 recognized as foreign 6 x 106 - Cap methylation reduces binding to pattern recognition receptors Raw Light Units 4 x 106 Pre-Clinical Manufacturing 2 x 106 2 - 3 Months Raw Material - Role of Cap 2 is largely unexplored Qualification 0 2 - 3 Months GMP Clinical wt wt Manufacturing 5moU 5moU 2 - 3 Months Product PseudoU PseudoU N1-Et-PUTPN1-FE-PUTPN1-Pr-PUTP N1-Et-PUTPN1-FE-PUTPN1-Pr-PUTP QC N1-Me-PUTP N1-Me-PUTP 1 Month N1-MOM-PUTP N1-MOM-PUTP Release and Ship • Primary sequence affects selection of optimal chemical 1-2 Weeks modifications • Uridine depletion of transcription template frequently improves incorporation by T7 RNA polymerase as well as transcription quality The Modified Nucleic Contact: Josh Whisenand Cap0: R1=H; R2=H • In numerous uridine-depleted sequence contexts, 5-methoxyuridine [email protected] OTS 2017 Cap1: R =CH ; R =H ® 1 3 2 yields high cell activity, low toxicity, and reduced interferon induction Acid Experts Bordeaux, FR 09/17 Cap2: R =CH ; R =CH 1 3 2 3 (Luciferase, Renilla, mCherry, ß-gal and others) www.trilinkbiotech.com.
Load more

Recommended publications
  • Active Site of the Mrna-Capping Enzyme Guanylyltransferase from Saccharomyces Cerevisiae
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6624-6628, July 1994 Biochemistry Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: Similarity to the nucleotidyl attachment motif of DNA and RNA ligases (nucleoidyl trnsfer/covalent catalysis) LUCILLE D. FRESCO* AND STEPHEN BURATOWSKI*t The Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142 Communicated by Phillip A. Sharp, March 8, 1994 ABSTRACT Nasent mRNAchains are capped atthe 5' end The covalent E-GMP intermediate (EnpG) can be conve- by the addiion ofa guanylyl residue to forma G(5')ppp(5')N... niently identified by radioactive labeling of the protein with structure. During the capping reaction, the guanylyltrnerase [a-32P]GTP. (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly Interest in the mechanism ofcapping has focused attention and covalently guanylylated. In this enzyme-GMP (E-GMP) on the E-GMP complex. Cellular mRNA guanylyltrans- termediate, GMP Is linked to the E-amino group of a lysine ferases from human, rat liver, calf thymus, Artemia salina, residue via a phosphoamide bond. Lys-70 was Identid as the wheat germ, and Saccharomyces cerevisiae all form E-GMP GMP attachment ste of the Saccharomyces cerevisiae guanylyl- covalent complexes (reviewed in ref. 3). In addition, cyto- transferase (encoded by the CEGI gene) by guanyylpeptide plasmic viruses encode their own capping enzymes. The sequencing. CEGI genes with substitutions at Lys-70 were guanylyltransferases of vaccinia virus (12, 13), reovirus (11, unable to support viability in yeast and produced proteins that 14, 15), African swine fever virus (16), rotavirus (17), blue- were not guanylylated in vitro.
    [Show full text]
  • RNA Capping Gene Expression in General Eukaryote Gene Expression Is Regulated at Seven Levels
    Modification and processing of eukaryotic pre-mRNAs RNA Capping Gene Expression in General Eukaryote gene expression is regulated at seven levels: 1. Transcription 2. RNA processing 3. mRNA transport 4. mRNA translation 5. mRNA degradation 6. Protein targeting 7. Protein degradation Each of these levels contains multiple steps, which are also subject of control and regulation Control vs. regulation The Long and Winding Road….. Processing of eukaryotic RNA polymerase II transcripts Steps in pre-mRNA processing i). Capping ii). Splicing iii). Cleavage and polyadenylation I. Capping II. Splicing a). Chemistry of mRNA splicing b). Donor and acceptor splice sites c). Spliceosome assembly and splice site recognition d). Small nuclear RNAs and RNPs e). Role of SR proteins in splicing f). Splicing regulation g). Alternative splicing h).Mutations that disrupt splicing i). AT-AC introns j). Trans splicing III. 3’ End Processing: Cleavage and Polyadenylation of Primary Transcripts Steps in mRNA processing (hnRNA is the precursor of mRNA) • capping (occurs co-transcriptionally) • cleavage and polyadenylation (forms the 3’ end) • splicing (occurs in the nucleus prior to transport) exon 1 intron 1 exon 2 Transcription of pre-mRNA and capping at the 5’ end cap Cleavage of the 3’ end and polyadenylation cap cap poly(A) Splicing to remove intron sequences cap poly(A) Transport of mature mRNA to the cytoplasm RNA capping 5’ cap 6 Most eukaryotic mRNAs contain a 5' cap of 7-methylguanosine 1 5 7 2 9 8 linked to the 5'-end of the mRNA in a novel 5', 5'-triphosphate linkage 3 4 This base is not encoded in the DNA template The cap may also have some additional modifications including: 1.Methyl groups at 2'-O position of the 1st and 2nd template encoded nucleotides No methyl at either 1st or 2nd template encoded nucleotides = Cap 0 Methyl at only 1st template encoded nucleotide = Cap 1 Methyl at both 1st and 2nd template encoded nucleotides = Cap 2 2.In some cases, if the 1st nucleotide is an adenine it may be methylated at N6 RNA capping Addition of the cap involves several step: 1.
    [Show full text]
  • 5'-Terminal Capping of RNA by Guanylyltransferase
    Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3758-3761, September 1977 Biochemistry 5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei (mRNA/7-methylguanosine/RNA processing) CHA-MER WEI AND BERNARD MOSS Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Robert P. Perry, June 24, 1977 ABSTRACT A soluble extract prepared from HeLa cell MATERIALS AND METHODS nuclei has been shown to catalyze the 5'-terminal modification Preparation of Guanylyltransferase from of RNA and synthetic polyribonucleotides to form m7G(5')pppA- HeLa Cells. and m7G(5')pppG- structures referred to as caps. The reaction HeLa S3 cells were harvested from suspension culture at 3 X involves the transfer of a GMP moiety from GTP to the 5' end 105 cells per ml and washed three times with 20mM Tris-HCl of an RNA molecule containing at Teast two terminal phos- (pH 7.6)/146 mM NaCl/11 mM glucose at 4°. The cells were phates. Significantly, neither the P nor the 'y phosphates of GTP then swollen in three volumes of 20mM Tris-HC1 (pH 7.9)/10 are transferred and polynucleotides with no 5'-terminal phos- mM MgCI2/10 mM KCI/0.25 mM spermidine at 00 and dis- phate or only one are not acceptors. In the absence of methyl rupted by Dounce homogenization. An equal volume of cold donor, G(5')pppA- and G(5')pppG- structures were synthesized, indicating that methylation is not required for guanylylation. 20mM Tris.HCI (pH 7.6)/50% glycerol was immediately added Cap formation was considered to occur by the following to the lysate.
    [Show full text]
  • Phosphorylation and Functions of the RNA Polymerase II CTD
    Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Phosphorylation and functions of the RNA polymerase II CTD Hemali P. Phatnani1 and Arno L. Greenleaf2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA The C-terminal repeat domain (CTD), an unusual exten- important that the heptads be in tandem: Insertion of an sion appended to the C terminus of the largest subunit of Ala residue between heptads is lethal in yeast, whereas RNA polymerase II, serves as a flexible binding scaffold insertion of an Ala between heptad pairs can be tolerated for numerous nuclear factors; which factors bind is de- (Stiller and Cook 2004). While the CTD is indispensable termined by the phosphorylation patterns on the CTD in vivo, it is frequently not required for general transcrip- repeats. Changes in phosphorylation patterns, as poly- tion factor (GTF)-mediated initiation and RNA synthesis merase transcribes a gene, are thought to orchestrate the in vitro (Zehring et al. 1988; Kim and Dahmus 1989; association of different sets of factors with the transcrip- Buratowski and Sharp 1990; Kang and Dahmus 1993; tase and strongly influence functional organization of Akoulitchev et al. 1995). Thus, the CTD does not form the nucleus. In this review we appraise what is known, part of the catalytic essence of RNAPII; rather, it must and what is not known, about patterns of phosphoryla- perform other functions. The nature and variety of these tion on the CTD of RNA polymerases II at the begin- functions are currently being elucidated and are a main ning, the middle, and the end of genes; the proposal that topic of this review.
    [Show full text]
  • Conventional and Unconventional Mechanisms for Capping Viral Mrna
    REVIEWS Conventional and unconventional mechanisms for capping viral mRNA Etienne Decroly1, François Ferron1, Julien Lescar1,2 and Bruno Canard1 Abstract | In the eukaryotic cell, capping of mRNA 5′ ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5′–3′ exonucleases and allows recognition of foreign RNAs (including viral transcripts) as ‘non-self’. However, viruses have evolved mechanisms to protect their RNA 5′ ends with either a covalently attached peptide or a cap moiety (7‑methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5′-caps and escape innate immunity. Pre-mRNA splicing The cap structure found at the 5′ end of eukaryotic reactions involved in the viral RNA-capping process, A post-transcriptional mRNAs consists of a 7‑methylguanosine (m7G) moi‑ and the specific cellular factors that trigger a response modification of pre-mRNA, in ety linked to the first nucleotide of the transcript via a from the innate immune system. which introns are excised and 5′–5′ triphosphate bridge1 (FIG. 1a). The cap has several exons are joined in order to form a translationally important biological roles, such as protecting mRNA Capping, decapping and turnover of host RNA functional, mature mRNA.
    [Show full text]
  • Reaction Mechanism of Mrna Guanylyltransferase from Rat Liver
    Proc. Nati Acad. Sci. USA Vol. 79, pp. 1693-1697, March 1982 Biochemistry Reaction mechanism of mRNA guanylyltransferase from rat liver: Isolation and characterization of a guanylyl-enzyme intermediate (capping mechanism/GTP-PP; exchange/covalent catalysis/phosphoamide linkage) KIYOHISA MIZUMOTOt, YOSHITo KAZIROt, AND FRITZ LIPMANNt tInstitute of Medical Science, University ofTokyo, Minatoku, Tokyo 108, Japan; and lThe Rockefeller University, New York, New York 10021 Contributed by Fritz Lipmann, October 27, 1981 ABSTRACT Rat liver RNA guanylyltransferase catalyzes a By studying the mechanism ofthe first reaction catalyzed by GTP-PP, exchange reaction in the absence of acceptor RNA [Mi- guanylyltransferase in detail, we found that a GTP-PPj exchange zumoto, K. & Lipmann, F. (1979) Proc. NatL Acai Sci USA 76, reaction occurred in the absence ofacceptor RNA and suggested 4961-4965] suggesting that the reaction proceeds through the for- that the guanylylation reaction may involve an enzyme-GMP mation of a covalent guanylylated intermediate. We now present complex as an intermediate (13). A similarfinding was also made more direct evidence for the existence of the enzyme-GMP in- by Shuman et al. (10) with the capping enzyme from vaccinia termediate: (i) the enzyme-[32P]GMP intermediate was formed virus. Furthermore, Shuman and Hurwitz (14) have recently on incubation of rat liver guanylyltransferase with [a-32P]GTP siicceed'ed' in isorating a covalent enzyme-GMP intermediate and migrated as a single radioactive band with Mr 69,000 on in which GMP is NaDodSO4polyacrylamide gel electrophoresis, and (ii) the in- linked to the Mr 95,000 subunit ofthe enzyme termediate isolated on gel filtration can transfer its GMP moiety complex.
    [Show full text]
  • Different Phosphorylated Forms of RNA Polymerase II and Associated Mrna Processing Factors During Transcription
    Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription Philip Komarnitsky,1 Eun-Jung Cho,1 and Stephen Buratowski2 Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts 02115, USA The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5؅ end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle. [Key Words: RNA polymerase II; TFIIH; capping enzyme; Kin 28] Received June 6, 2000; revised version accepted July 31, 2000. Eukaryotic mRNAs undergo 5Ј end capping, splicing of ase. In contrast, the mRNA cap methyltransferase Abd1 introns, and polyadenylation.
    [Show full text]
  • 5 -Capping Enzymes Are Targeted to Pre-Mrna by Binding to The
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press 5*-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II Susan McCracken, Nova Fong, Emanuel Rosonina,1 Krassimir Yankulov, Greg Brothers, David Siderovski, Andrew Hessel, Stephen Foster,2 Amgen EST Program,2 Stewart Shuman,3 and David L. Bentley1,4 Amgen Institute and Ontario Cancer Institute, Toronto, Ontario M5G 2C1, Canada; 1Department of Medical Biophysics, University of Toronto, Toronto, Canada; 2 Amgen, Inc., Thousand Oaks, California 91320 USA; 3Memorial Sloan-Kettering Cancer Center, New York, New York 10021 USA We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5* capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5* ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation. [Key Words: RNA Pol II; CTD; mRNA cap; RNA guanylyltransferase; RNA 7-methyltransferase] Received September 22, 1997; accepted in revised form October 14, 1997. Transcripts made by RNA Polymerase II (Pol II) are dis- subunits copurify as a stable complex distinct from the tinguished from Pol I and Pol III transcripts by a m7G(58)- monomeric methyltransferase (Itoh et al.
    [Show full text]
  • Origins of Mrna Identity: Capping Enzymes Bind to The
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12758–12760, November 1997 Commentary Origins of mRNA identity: Capping enzymes bind to the phosphorylated C-terminal domain of RNA polymerase II Stewart Shuman Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021 Cellular enzymes that cap, splice, and polyadenylate eukary- Cap formation entails a series of three reactions catalyzed by otic pre-mRNAs are targeted in vivo to the nascent chains RNA triphosphatase, RNA guanylyltransferase, and RNA synthesized by RNA polymerase II (pol II). Placing a mam- (guanine-7-) methyltransferase. The triphosphatase hydro- malian pol II transcription unit under the control of a pol III lyzes the 59 triphosphate end of the primary transcript to a promoter results in a failure to cap, splice, or polyadenylate the diphosphate. Guanylyltransferase adds GMP from GTP to the transcript (1, 2). How is pre-mRNA ‘‘identity’’ established? Do diphosphate RNA end to form a blocked G(59)ppp(59)N the various mRNA processing enzymes recognize protein structure. The methyltransferase adds a methyl group from components of the pol II transcription elongation complex or S-adenosylmethionine to the cap guanosine to form is identity conferred through an initial pol II-specific modifi- m7GpppN. This pathway was elucidated more than 20 years cation that directs subsequent mRNA fate? We know that ago by the Moss and Shatkin laboratories through studies of acquisition of the m7GpppN cap is the first modification event the capping enzymes encoded by vaccinia virus and reovirus in mRNA biogenesis and that capping facilitates downstream (reviewed in ref. 14). Only in the past few years have cellular transactions such as splicing, polyadenylation, transport, and genes encoding the capping enzymes been cloned (15–19).
    [Show full text]
  • High Resolution Crystal Structure of a Key Editosome Enzyme from Trypanosoma Brucei: RNA Editing Ligase 1
    doi:10.1016/j.jmb.2004.08.041 J. Mol. Biol. (2004) 343, 601–613 High Resolution Crystal Structure of a Key Editosome Enzyme from Trypanosoma brucei: RNA Editing Ligase 1 Junpeng Deng1,2, Achim Schnaufer3,4, Reza Salavati3,4 Kenneth D. Stuart3,4 and Wim G. J. Hol1,2* 1Howard Hughes Medical Trypanosomatids are causative agents of several devastating tropical diseases Institute, University of such as African sleeping sickness, Chagas’ disease and leishmaniasis. There Washington, Seattle WA 98195 are no effective vaccines available to date for treatment of these protozoan USA diseases, while current drugs have limited efficacy, significant toxicity and suffer from increasing resistance. Trypanosomatids have several remarkable 2Department of Biochemistry and unique metabolic and structural features that are of great interest for and Biological Structure developing new anti-protozoan therapeutics. One such feature is “RNA Biomolecular Structure Center editing”, an essential process in these pathogenic protozoa. Transcripts for key University of Washington trypanosomatid mitochondrial proteins undergo extensive post-transcrip- P.O. Box 357742, Seattle WA tional RNA editing by specifically inserting or deleting uridylates from pre- 98195, USA mature mRNA in order to create mature mRNAs that encode functional 3Seattle Biomedical Research proteins. The RNA editing process is carried out in a w1.6 MDa multi-protein Institute, 307 Westlake Avenue complex, the editosome. In Trypanosoma brucei, one of the editosome’s core N, Suite 500, Seattle, WA 98109 enzymes, the RNA editing ligase 1 (TbREL1), has been shown to be essential USA for survival of both insect and bloodstream forms of the parasite. We report ˚ 4 here the crystal structure of the catalytic domain of TbREL1 at 1.2 A Department of Pathobiology resolution, in complex with ATP and magnesium.
    [Show full text]
  • Curriculum Vitae Stewart Shuman, MD, Ph.D. Address
    Curriculum Vitae Stewart Shuman, M.D., Ph.D. Address: Molecular Biology Program Sloan-Kettering Institute 1275 York Ave. New York, NY 10065 [email protected] Education: B.A. Biology (1976) summa cum laude Wesleyan University Middletown, Connecticut Ph.D. Molecular Biology (1983) Albert Einstein College of Medicine, New York M.D. (1983) Albert Einstein College of Medicine Postgraduate Positions Held: 1983-86 Medical House Officer, Medical Services Massachusetts General Hospital 1986-88 Medical Staff Fellow, Laboratory of Viral Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health 1988-92 Assistant Member, Molecular Biology Program, Sloan-Kettering Institute, and Assistant Member, Dept. of Medicine, Memorial Sloan-Kettering Cancer Center 1992-95 Associate Member, Molecular Biology Program, Sloan-Kettering Institute, and Associate Member, Dept. of Medicine, Memorial Sloan-Kettering Cancer Center 1995- Member, Molecular Biology Program, Sloan-Kettering Institute, and Member, Dept. of Medicine, Memorial Sloan-Kettering Cancer Center Certification: American Board of Internal Medicine (1986) Honors: Member of the American Academy of Arts and Sciences (2015) Fellow of the American Academy of Microbiology (2013) NIH MERIT Award (2007-2017) American Cancer Society Research Professor (2005 - ) American Society for Virology, Wolfgang Joklik Lectureship (2004) Burroughs Wellcome Fund Award - New Initiatives in Malaria Research (2001-03) Simon H. Rifkind Chair (1999 - ) American Cancer Society Faculty Research
    [Show full text]
  • Highly Efficient 5' Capping of Mitochondrial RNA with NAD+ and NADH by Yeast and Human Mitochondrial RNA Polymerase
    Thomas Jefferson University Jefferson Digital Commons Department of Biochemistry and Molecular Department of Biochemistry and Molecular Biology Faculty Papers Biology 12-12-2018 Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase Jeremy G Bird Rutgers University Urmimala Basu Rutgers University David Kuster Rutgers University; Heidelberg University Aparna Ramachandran FRutgersollow this Univ andersity additional works at: https://jdc.jefferson.edu/bmpfp Ewa P arGrudzien-Nogalskat of the Medical Biochemistr y Commons LetRutgers us Univ knowersity how access to this document benefits ouy SeeRecommended next page for Citation additional authors Bird, Jeremy G; Basu, Urmimala; Kuster, David; Ramachandran, Aparna; Grudzien-Nogalska, Ewa; Towheed, Atif; Wallace, Douglas C.; Kiledjian, Megerditch; Temiakov, Dmitry; Patel, Smita S.; Ebright, Richard H.; and Nickels, Bryce E., "Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase" (2018). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 144. https://jdc.jefferson.edu/bmpfp/144 This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Department of Biochemistry and Molecular Biology Faculty Papers by an authorized administrator of the Jefferson Digital Commons.
    [Show full text]