Metabolism of Nuclear RNA
Coding and non-coding RNAzzCoding RNAs (4% ) - transcriptomemRNAs : rapid turnoverhalf-life - few minutes (bacteria) few hours(eukaryotes)Non-coding RNAs (96% )rRNAs : 80% in actively dividing bacteriatRNAssnRNAs : included in mRNA processing (spliceosome),eukaryotes onlysnoRNAs : included in rRNA processing, eukaryotesonlymi-RNAs: RNA-induced transcription silencingRNA processingEnd-modifications : 7-methylguanosine cap, poly(A) tailSplicing : removal of intronsCutting events : rRNA, tRNA processingChemical modifications : addition of chemicalgroups, RNA editing
RNA Pol II is an RNA FactoryCapping of RNA pol IItranscriptszzzoccurs early in all RNA pol IItranscripts when the nascent RNAis in 20-35 nt stage (hnRNA, presnRNA)after cleaving the γ-phosphate(RNA-triphosphatase) a GMP isadded in inverted orientation (5 - 5 linkage) (RNAguanylyltranspherase)Terminal inverted guanosine ismethylated at the 7 position (7metyltranspherase)
Functions of CapzProtects mRNA from degradation by RNAseszEnhances splicing efficiency of mRNAszEnhances transport of mRNAs from nucleus to cytoplasmzIn the cytoplasm, the cap-structure is important for the initiation oftranslationThe eukaryotic translation initiation factor eIF4E binds directly to thecap-structuresnRNAs m7G - nuclear export, m3G - nuclear importzzPolyadenylationzzzzzzzRequires the sequence 5'-AAUAAA-3' plusthe sequence 5'-CA-3' 10-20 nt downstream, plusa 5'-(GU)-3' rich sequence further downstreamSix factors are needed (PAP, CPSF, CSF, CFI, CFII, PAB),Two stage reaction (cleavage and AMP addition)100-200 A residues added to give ‘poly(A) tail’mRNA encoding histones are not polyadenylated
Main steps in icity factor) binds AAUAAA inhnRNAz CStF (cleavage stimulatory factor)binds G/U rich sequence; CFI, CFIIbind in betweenz PAP (PolyA polymerase) binds &cleaves 10-35 b 3' to AAUAAA at slowratez PAP adds about 12 A residues slowly,CFI, CFII and CPSF fall offzzPABII (polyA-binding protein II)binds, now As are added rapidly untilreach 250, then stopsPurpose of PolyadenylationzzzzProtects mRNA – In cytoplasm poly(A) size decreases due toRNAses, however poly(A) polymerase continues rebuildingNecessary for translation (site of binding by poly(A)-binding protein I(PABI)Recruits mRNA to polysomesRequired for splicing out of the last intron
pre-mRNA splicingzz1.2.3.Introns are intervening sequences that “interrupt” eukaryotic genesand must be removed before uninterrupted exons coding forproteins leave the nucleus as mRNAThree types of intron are known:Group I introns - Found in organelle and bacterial genes along withsome lower eukaryotes (fungi, tetrahymena) nuclear genes Can self splice without the aid of proteins Require free GTP for splicingGroup II introns - Found in organelle and bacterial genes Can self splice without the aid of proteins Differ from Group I introns in sequence and mechanismNuclear introns - Found in eukaryotic nuclear genes Require proteins and other RNAs for splicingSelf splicing intronszzzzzS.Altmann and T. Cechgroup I requires binding of guanosine into active siteresults of splicing are mature RNA and linear intron RNAgroup II does not bind any nucleotide it uses activatedOH of adenosine in active in branch site.results of splicing are mature RNA and lariat intron
Splicing of nuclear intronszzzzzzRequires spliceosomeSpliceosome consists of snRNPs and at least 70 participatingproteinsTransesterifications are facilitated by base-pairing of snRNAsMost mRNA are derived from the corresponding pre-mRNATrypanosomas and Euglenoids construct mRNA from separateprecursors – trans splicingStandard intron boundary – ‘GU-AG’ rule
SR Proteins Direct Splicingby Defining ExonszzzzSR proteins family of 8 well conserved proteins.SR proteins have N-terminal RNP domains rich in serand arg.Function to align 5’ splice site and the branch pointby interactions with snRNP U1 and snRNP U2.Regulators of alternative splicing:
Alternative splicingWalter Gilbert - 1977z 40 - 60% of pre-mRNA transcriptsundergoes alternative splicingz Allow for tissue specific expressionz Immunoglobin genes, Drosophila sexdeterminationzTransport of mRNA: Balbiani rings
mRNA export and its alternate fates in the cytoplasmWhat is RNA Interference(RNAi)“The Process by which dsRNA silencesgene expression.” [Mittal, 2004]z Generally: Post transcriptional genesilencing (PTGS)z Results in : Degradation or translationinhibition: Heterochromatin formationz A field with many unknownsz
Handy RNAi TermsdsRNA: double stranded RNA, longerthan 30 ntz miRNA: microRNA, 21-25 nt.zz Encodedby endogenous genes.z Hairpin precursorsz Recognize multiple targets.zsiRNA: short-interfering RNA, 21-25 nt.z Mostlyexogenous origin.z dsRNA precursorsz May be target specificRNAi: Two Phase ProcesszInitiationz Generationzof mature siRNA or miRNAExecutionz Silencingof target genez Degradation or inhibition of translation
miRNA BiogenesiszzzTranscribed from endogenous gene as pri-miRNAz Primary miRNA: long with multiple hairpinsz Imperfect internal sequence complementarityCleaved by Drosha into pre-miRNAz Precursor miRNA: 70nt imperfect hairpinsz Exported from nucleusCleaved by Dicer into mature miRNAz 21-25ntz Symmetric 2nt 3’ overhangs, 5’ phosphate groupsNovina and Sharp, 2004siRNA BiogenesisDicer cleaves long dsRNA into siRNA 2125ntz dsRNA from exogenous sourcesz Symmetric 2nt 3’ overhangs, 5’phosphate groupsz Evidence for amplification in C. elegansand plantsz Allows persistence of RNAi?zNovina and Sharp, 2004
Protein machinery of RNAizDrosha - Processes pri-miRNA into pre-miRNA- Leaves 3’ overhangs on pre-miRNAzDicer- Cleaves dsRNA or pre-miRNA- Leaves 3’ overhangs and 5’ phosphategroupszRNA Dependent RNA Polymerase (RdRP) siRNA acts as primer for elongation ontarget mRNA – more dsRNA producedzArgonaute (Ago) - lies at the heart all RNAzRNA Induced Silencing Complex (RISC) - RNAizRNA induced transcriptional silencing (RITS) - RISC-silencing effector complexeseffector complexlike complex responsible for heterochromatin formation inS.pombe.
Proposed Biologic Roles‘Immune System’ of the Genome Antiviral Defense Suppress Transposon Activity Gene Regulation (Silencing)(e.g. MicroRNAs, Heterochromatin)RNAi ApplicationsGENETIC TOOLProbing Gene FunctionGENE THERAPYCombat Viral InfectionTreat Genetic Diseases(New expression strategies)
Setting up an RNAi experimentdesign of RNA oligosz siRNA synthesisz Introducing siRNA into the cellsz monitoring effectszDesign of specific siRNAzzzzMultiple siRNA to single targetConsider: Length: usually 19-23 nt (27-mers Kim et al. Nature Biotechnology, 2005),Content of G/C: (30-52%),Positions of A/U: in the sense strandPosition in target mRNA: at least 100bases downstream AUG,Avoid 5 or 3 UTR (Boese et al.,Methods in enzymology, 2005).Scrambled controlPositive control (GAPDH)
siRNA synthesiszChemical synthesiszIn vitro transcriptionzDigestion of long dsRNA by an RNase III family enzyme(e.g. Dicer, RNase III)zExpression in cells from an siRNA expression plasmid orviral vectorzExpression in cells from a PCR-derived siRNAexpression cassetteIntroducing siRNA into the cellsTransfectionz Electroporationz Concentration ranging from 5 to 50 nMz
Monitoring effectsqReal-Time PCRz Western blotz Immunofluorescencez Monitoring of interferon response: 2'5'oligoadenylate synthetase, dsRNAdependent protein kinase, PKRzThank you.
Coding and non-coding RNA zCoding RNAs (4% ) - transcriptome mRNAs : rapid turnover . RNA editing . RNA Pol II is an RNA Factory Capping of RNA pol II transcripts . Methods in enzymology, 2005). zScrambled control zPositive control (GAPDH) siRNA synthesis
(Structure of RNA from Life Sciences for all, Grade 12, Figure 4.14, Page 193) Types of RNA RNA is manufactured by DNA. There are three types of RNA. The three types of RNA: 1. Messenger RNA (mRNA). It carries information about the amino acid sequence of a particular protein from the DNA in the nucleus to th
The process of protein synthesis can be divided into 2 stages: transcription and translation. 5 as a template to make 3 types of RNA: a) messengermessenger--RNA (mRNA)RNA (mRNA) b) ribosomalribosomal--RNA (rRNA)RNA (rRNA) c) transfertransfer--RNA (tRNA)RNA (tRNA) Objective 32 2)2) During During translationtranslation, the
10 - RNA Modiﬁcations After the RNA molecule is produced by transcription (Part 9), the structure of the RNA is often modified prior to being translated into a protein. These modifications to the RNA molecule are called RNA modifications or posttranscriptional modifications. Most RNA modifications apply onl
13.1 RNA RNA Synthesis In transcription, RNA polymerase separates the two DNA strands. RNA then uses one strand as a template to make a complementary strand of RNA. RNA contains the nucleotide uracil instead of the nucleotide thymine. Follow the direction
DNA AND RNA Table 4.1: Some important types of RNA. Name Abbreviation Function Messenger RNA mRNA Carries the message from the DNA to the protein factory Ribosomal RNA rRNA Comprises part of the protein factory Transfer RNA tRNA Transfers the correct building block to the nascent protein Interference RNA
Nuclear Chemistry What we will learn: Nature of nuclear reactions Nuclear stability Nuclear radioactivity Nuclear transmutation Nuclear fission Nuclear fusion Uses of isotopes Biological effects of radiation. GCh23-2 Nuclear Reactions Reactions involving changes in nucleus Particle Symbol Mass Charge
Biological Functions of Nucleic Acids tRNA (transfer RNA, adaptor in translation) rRNA (ribosomal RNA, component of ribosome) snRNA (small nuclear RNA, component of splicesome) snoRNA (small nucleolar RNA, takes part in processing of rRNA) RNase P (ribozyme, processes tRNA) SRP RNA (
4 Eukaryotic genes 1) ribosomal RNA (rRNA) - 4 ribosomal RNA genes from 2 transcripts 2) transfer RNA (tRNA) - carry amino acids that are incorporated into proteins during translation. 3) messenger RNA (mRNA) - translated into proteins 4) heterogeneous nuclear RNA (hnRNA) - an umbrella term that encompases a
A summary of transcription and translation in a eukaryotic cell TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase RNA transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cyt
Cell Biology and Enzymology Prokaryotic and eukaryotic cells, cell wall, nucleus, mitochondria, Golgi bodies, lysosomes, . RNA polymerases, capping, elongation, and termination, RNA processing, RNA editing, splicing, and polyadenylation, structure and function of different types of RNA, RNA transport). . Methods of microbial gene transfer .
A) Metabolism depends on a constant supply of energy from food B) Metabolism depends on an organismʹs adequate hydration C) Metabolism utilizes all of an organismʹs resources D) Metabolism is a property of organismal life E) Metabolism manages the increase of entropy in an organism Answer: D Topic: Concepts 8.1, 8.5
The nucleus is the manager of the cell THE NUCLEUS AND RIBOSOMES: GENETIC CONTROL OF THE CELL Genes found on the chromosomes within the nucleus store information necessary to produce proteins. The nucleus is bordered by a double membrane called the nuclear envelope Structure and Function of the Nucleus It contains chromatin It
Guide for Nuclear Medicine NUCLEAR REGULATORY COMMISSION REGULATION OF NUCLEAR MEDICINE. Jeffry A. Siegel, PhD Society of Nuclear Medicine 1850 Samuel Morse Drive Reston, Virginia 20190 www.snm.org Diagnostic Nuclear Medicine Guide for NUCLEAR REGULATORY COMMISSION REGULATION OF NUCLEAR MEDICINE. Abstract This reference manual is designed to assist nuclear medicine professionals in .
Bohr’s model of the atom is still a convenient description of the energy levels of the hydrogen atom. Fig. 2-2. The nucleus. Chapter 2—The Atomic Nucleus 2-3 The Nucleus The nucleus depicted in Fig. 2-2 is now understood to be a quantum system composed of protons and neutrons, particles of nearly equal mass and the same intrinsic .
Two years later, James Chadwick and E.S. Bieler conclude that some strong force holds the nucleus together. Why? If protons in nucleus (and not electrons) then what keeps the repulsive force from driving the nucleus apart? Nucleus confined to a very small volume ( 10-14 m in d
the nucleus is not attracted by the electrons; the nucleus attracts an electron more than the electron attracts the nucleus; the protons in the nucleus attract one electron each; and the electrons repel the nucleus. A . classroom probe designed to elicit these, and related ideas, from post-1
Nucleus (double membrane) Contains genetic information within DNA of chromosomes of the cell; provides the template (RNA) for protein formation. Controls activities of the cell Nucleolus Made of protein and RNA and is in the nucleus. Synthesises and assembles ribosomes which leave the nucleus and enter the cytoplasm Mitochondria (double membrane)
Transcription To make protein from nucleotide code of RNA copy of chromosome DNA RNA strand copy binds with DNA strand Messenger RNA (mRNA) Leaves nucleus Shuttles between nucleus and cytoplasma of cell
From Fields Virology Schematic of Replication Cycle of ( ) RNA Single Strand Viruses Coding for Genomic and Subgenomic RNA’s Genomic RNA binds to ribosomes but only a portion of 5’ end is translated into non-structural proteins (-) strand RNA is synthesized. Different classes of ( ) RNA’s are produced. One is trans-lated into a .
15th AMC ! 8 1999 5 Problems 17, 18, and 19 refer to the following: Cookies For a Crowd At Central Middle School the 108 students who take the AMC! 8 meet in the evening to talk about prob-lems and eat an average of two cookies apiece. Walter and Gretel are baking Bonnie’s Best Bar Cookies this year. Their recipe, which makes a pan of 15 cookies, list these items: 11 2 cups of our, 2 eggs .