Transcription and Post-transcription Modification Post-transcriptional Processing of RNAMaking ends of RNARNA splicingPrimaryTranscriptPrimary Transcript-the initial molecule of RNA produced- hnRNA （heterogenous nuclear RNA ）In prokaryotes, DNA RNA protein in cytoplasm concurrently In eukaryotes nuclear RNA Cp RNA Processingofeukaryoticpre-mRNAHumandystrophingenehas79exons,spansover2,300-Kbandrequiresover16hourstobetranscribed!Forprimarytranscriptscontainingmultipleexonsandintrons,splicingoccursbeforetranscriptionofthegeneiscomplete-co-transcriptionalsplicing.Types of RNA processingA) Cutting and trimming to generate ends:rRNA, tRNA and mRNAB) Covalent modification:Add a cap and a polyA tail to mRNAAdd a methyl group to 2-OH of ribose in mRNA and rRNAExtensive changes of bases in tRNAC) Splicingpre-rRNA, pre-mRNA, pre-tRNA by different mechanisms.TheRNAPolIICTDisrequiredforthecouplingoftranscriptionwithmRNAcapping,polyadenylationandsplicing1.Thecouplingallowstheprocessingfactorstopresentathighlocalconcentrationswhensplicesitesandpoly(A)signalsaretranscribedbyPolII,enhancingtherateandspecificityofRNAprocessing.2.TheassociationofsplicingfactorswithphosphorylatedCTDalsostimulatesPolIIelongation.Thus,apre-mRNAisnotsynthesizedunlessthemachineryforprocessingitisproperlypositioned. Time course of RNA processing 5 and 3 ends of eukaryotic mRNAAdd a GMPMethylate it and1st few nucleotidesCut the pre-mRNAand add As5-UTR3-UTRCappingofpre-mRNAsCap=modified guanine nucleotideCapping= first mRNA processing event - occurs during transcriptionCTD recruits capping enzyme as soon as it is phosphorylatedPre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bp longCap structure is recognized by CBC（cap-binding complex） stablize the transcript prevent degradation by exonucleases stimulate splicing and processingSometimesmethylatedSometimesmethylatedThecapisaddedafterthenascentRNAmoleculesproducedbyRNApolymeraseIIreachalengthof25-30nucleotides.GuanylyltransferaseisrecruitedandactivatedthroughbindingtotheSer5-phosphorylatedPolIICTD.ThemethylgroupsarederivedfromS-adenosylmethionine.CappinghelpsstabilizemRNAandenhancestranslation,splicingandexportintothecytoplasm.Capping of the 5 end of nascent RNA transcripts with m7GExistinginasinglecomplexConsensus sequence for 3 processAAUAAA: CstF (cleavage stimulation factor F)GU-rich sequence: CPSF (cleavage and polyadenylation specificity factor)Polyadenylation of mRNA at the 3 endCPSF: cleavage and polyadenylation specificity factor.CStF: cleavage stimulatory factor.CFI & CFII: cleavage factor I & II.PAP: poly(A) polymerase.PABPII: poly(A)-binding protein II.Poly(A) tail stabilizes mRNA and enhances translation and export into the cytoplasm.RNA is cleaved 1035-nt 3 to A2UA3.The binding of PAP prior to cleavage ensures that the free 3 end generated is rapidly polyadenylated.PAP adds the first 12A residues to 3-OH slowly.Binding of PABPII to the initial short poly(A) tail accelerates polyadenylation by PAP.The polyadenylation complex is associated with the CTD of Pol II following initiation.Functions of 5 cap and 3 polyANeed 5 cap for efficient translation:Eukaryotic translation initiation factor 4 (eIF4) recognizes and binds to the cap as part of initiation.Both cap and polyA contribute to stability of mRNA:Most mRNAs without a cap or polyA are degraded rapidly.Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast.mRNA Half-life t seconds if seldom needed t several cell generations (i.e. 48-72 h) for houskeeping gene avg 3 h in eukaryotes avg 1.5 min in bacteria Poly(A)+ RNA can be separated from other RNAs by fractionation on Sepharose-oligo(dT). Split gene and mRNA splicingSITEFEEDBACKCONTACTTELLAFRIENDLastmodifiedMay10,2001TheOfficialWebSiteofTheNobelFoundationCopyright2004TheNobelFoundationBackground: Adenovirus has a DNA genome andmakes many mRNAs. Can we determine whichpart of the genome encodes for each mRNA bymaking a DNA:RNA hybrid?Experiment: Isolate Adenovirus genomic DNA, isolate one adenovirus mRNA, hybridize and then look by EM at where the RNA hybridizes (binds) to the genomic DNA.Surprise: The RNA is generated from 4 different regions of the DNA! How can weexplain this? Splicing! Thediscoveryofsplitgenes(1977)1993NoblePrizeinMedicineToDr.RichardRobertandDr.PhillipSharpThematuredmRNAsaremuchshorterthantheDNAtemplates.DNAmRNAExon and Intron Exon is any segment of an interrupted gene that is represented in the mature RNA product. Intron is a segment of DNA that is transcribed, but removed from within the transcript by splicing together the sequences (exons) on either side of it.ExonsaresimilarinsizeIntronsarehighlyvariableinsizeGT-AGruleGT-AGruledescribesthepresenceoftheseconstantdinucleotidesatthefirsttwoandlasttwopositionsofintronsofnucleargenes.Splicesitesarethesequencesimmediatelysurroundingtheexon-intronboundariesSplicingjunctionsarerecognizedonlyinthecorrectpairwisecombinationsThe sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.The consensus sequence at the exonintron junctions of vertebrate pre-mRNAs.4 major types of introns 4 classes of introns can be distinguished on the basis of their mechanism of splicing and/or characterisitic sequences:Group I introns in fungal mitochondria, plastids, and in pre-rRNA in Tetrahymena (self-splicing)Group II introns in fungal mitochondria and plastids (self-splicing)Introns in pre-mRNA (spliceosome mediated) Introns in pre-tRNAGroup I and II intronsThesequenceoftransesterificationreactionsthatsplicetogethertheexonsofeukaryoticpre-mRNAs.Splicing of Group I and II intronsIntrons in fungal mitochondria, plastids, Tetrahymena pre-rRNAGroup ISelf-splicingInitiate splicing with a G nucleotideUses a phosphoester transfer mechanism Does not require ATP hydrolysis.Group IIself-splicingInitiate splicing with an internalAUses a phosphoester transfer mechanismDoes not require ATP hydrolysisSelf-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981Exon1Exon2Intron1Exon1 Exon2Intron1+pre-rRNASplicedexonIntroncircleIntronlinearpre-rRNANuclearextractGTP+-+-+-+-Productsofsplicingwereresolvedbygelelectrophoresis:AdditionalproteinsareNOTneededforsplicingofthispre-rRNA!DoneedaGnucleotide(GMP,GDP,GTPorGuanosine).Thesequenceofreactionsintheself-splicingofTetrahymenagroupIintron.Where is the catalytic activity in RNase P?RNase P is composed of a 375 nucleotide RNA and a 20 kDa protein. The protein component will NOT catalyze cleavage on its own.The RNA WILL catalyze cleavage by itself !The protein component aids in the reaction but is not required for catalysis.ThusRNA can be an enzyme.Enzymes composed of RNA are called ribozymes.Hammerhead ribozymesA 58 nt structure is used in self-cleavageThe sequence CUGA adjacent to stem-loops is sufficient for cleavage Mechanism of hammerhead ribozymeThe folded RNA forms an active site for binding a metal hydroxideAttracts a proton from the 2 OH of the nucleotide at the cleavage site. This is now a nucleophile for attack on the 3 phosphate and cleavage of the phosphodiester bond.1989 Nobel Prize in chemistry, Sidney Altman, and Thomas Cech DistributionofGroupIintronsProkaryotes eubacteria (tRNA & rRNA), phageEukaryoteslower (algae, protists, & fungi)nuclear rRNA genes, organellar genes, Chlorella viruseshigher plants: organellar geneslower animals (Anthozoans): mitochondrial1800 known, classified into 12 subgroups, based on secondary structureSplicing of pre-mRNAThe introns begin and end with almost invariant sequences: 5 GUAG 3Use ATP hydrolysis to assemble a large spliceosome (45S particle, 5 snRNAs and 65 proteins, same size and complexity as ribosome)Mechanism is similar to that of the Group II fungal introns:Initiate splicing with an internal AUses a phosphoester transfer mechanism for splicingInitiation of phosphoester transfers in pre-mRNAUses 2 OH of an A internal to the intronForms a branchpoint by attacking the 5 phosphate on the first nucleotide of the intronForms a lariat structure in the intronExons are joined and intron is excised as a lariatA debranching enzyme cleaves the lariat at the branch to generate a linear intronLinear intron is degradedInvolvement of snRNAs and snRNPssnRNAs = small nuclear RNAssnRNPs = small nuclear ribonucleoproteins particles (snRNA complex with protein)Addition of these antibodies to an in vitro pre-mRNA splicing reaction blocked splicing.Thus the snRNPs were implicated in splicingRecognizingthe 5 splice site and the branch site.Bringingthose sites together.Catalyzing(or helping to catalyze) the RNA cleavage.Role of snRNPs in RNA splicingRNA-RNA,RNA-proteinandprotein-proteininteractionsareallimportantduringsplicingsnRNPs U1, U2, U4/U6, and U5 snRNPsHave snRNA in each: U1, U2, U4/U6, U5Conserved from yeast to humanAssemble into spliceosomeCatalyze splicingSplicingofpre-mRNAoccursina“spliceosome”anRNA-proteincomplexpre-mRNAsplicedmRNAspliceosome(100proteins+5smallRNAs)Thespliceosomeisalargeprotein-RNAcomplexinwhichsplicingofpre-mRNAsoccurs.Assembly of spliceosomesnRNPs are assembled progressively into the spliceosome.U1 snRNP binds (and base pairs) to the 5 splice siteBBP (branch-point binding protein) binds to the branch siteU2 snRNP binds (and base pairs) to the branch point, BBP dissociatesU4U5U6 snRNP binds, and U1 snRNP dissociatesU4 snRNP dissociatesAssembly requires ATP hydrolysisAssembly is aided by various auxiliary factors and splicing factors.Some RNA-RNA hybrids formed during the splicing reactionSteps of the spliceosome-mediated splicing reactionAschematicdiagramofsixrearrangementsthatthespliceosomeundergoesinmediatingthefirsttransesterificationreactioninpre-mRNAsplicing.Assembly of spliceosomeThe spliceosome cycleThe Significance of Gene SplicingThe introns are rare in prokaryotic structural genesThe introns are uncommon in lower eukaryote (yeast), 239 introns in 6000 genes, only one intron / polypeptideThe introns are abundant in higher eukaryotes (lacking introns are histons and interferons)Unexpressed sequences constitute 80% of a typical vertebrate structural gene Errors produced by mistakes in splice-site selectionMechanisms prevent splicing errorCo-transcriptional loading processSR proteins recruit spliceosome components to the 5 and 3 splice sitesSR protein = Serine Arginine rich proteinESE = exonic splicing enhancersSR protein regulates alternative splicingAlternative splicingAlternative splicing occurs in all metazoa and is especially prevalent in vertebrateFivewaystospliceanRNARegulated alternative splicingDifferentsignalsinthepre-mRNAanddifferentproteinscausespliceosomestoforminparticularpositionstogivealternativesplicing76575657Faspre-mRNAAPOPTOSISAlternativesplicingcangeneratemRNAsencodingproteinswithdifferent,evenoppositefunctions(programmedcelldeath)FasligandSolubleFas(membrane)FasFasligand(membrane-associated)(+)(-) Alternative possibilities for 4 exons leave a total number of possible mRNA variations at 38,016. The protein variants are important for wiring of the nervous system and for immune response.DrosophilaDscamgenecontainsthousandsofpossiblesplicevariantsCis- and Trans-SplicingCis-: Splicing in single RNATrans-: Splicing in two different RNAs Y-shaped excised introns (cis-: lariat) Occur in C. elegance and higher eukaryotes but it does in only a few mRNAs and at a very low level pre-mRNAsplicingtrans-mRNAsplicingsplicedleaderSamesplicingmechanismisemployedintrans-splicingSplicedleadercontainsthecapstructure!RNA editingRNA editing is the process of changing the sequence of RNA after transcription.In some RNAs, as much as 55% of the nucleotide sequence is not encoded in the (primary) gene, but is added after transcription.Examples: mitochondrial genes in Trypanosomes （锥虫）Can add, delete or change nucleotides by editingTwo mechanisms mediate editingGuideRNA-directeduridineinsertionordeletionSite-specificdeaminationInsertion and deletion of nucleotides by editingUses a guideRNA (in 20S RNP = editosome) that is encoded elsewhere in the genomePart of the guide RNA is complementary to the mRNA in vicinity of editing TrypanosomalRNAeditingpathways.(a)Insertion.(b)Deletion. Mammalian example of editingThe C is converted to U in intestine by a specific deaminating enzyme, not by a guide RNA.Cutting and Trimming RNACan use endonucleases to cut at specific sites within a longer precursor RNACan use exonucleases to trim back from the new ends to make the mature productThis general process is seen in prokaryotes and eukaryotes for all types of RNAThe posttranscriptional processing of E. coli rRNA.RNase III cuts in stems of stem-loops16S rRNA23S rRNARNase IIINo apparent primary sequence specificity - perhaps RNase III recognizes a particular stem structure.Eukaryotic rRNA ProcessingThe primary rRNA transcript (7500nt, 45S RNA) contains 18S, 5.8S and 28SMethylationoccur mostly in rRNA sequence80%: O2-methylribose, 20%: bases (A or G) peudouridine 95 U in rRNA in human are converted to Ysmay contribute rRNA tertiary stabilityTransfer RNA ProcessingCloverleaf structureCCA: amino acid binding siteAnticodon60 tRNA genes in E. coliAschematicdiagramofthetRNAcloverleafsecondarystructure.Endo- and exonucleases to generate ends of tRNAEndonuclease RNaseP cleaves to generate the 5 end.Endonuclease RNaseF cleaves 3 nucleotides past the mature 3 end.Exonuclease RNaseD trims 3 to 5, leaving the mature 3 end.Splicing of pre-tRNAIntrons in pre-tRNA are very short (about 10-20 nucleotides)Have no consensus sequencesAre removed by a series of enzymatic steps:Cleavage by an endonucleasePhosphodiesterase to open a cyclic intermediate and provide a 3OHActivation of one end by a kinase (with ATP hydrolysis)Ligation of the ends (with ATP hydrolysis)Phosphatase to remove the extra phosphate on the 2OH (remaining after phosphodiesterase )Steps in splicing of pre-tRNAPOH52,3cyclicphosphateExcisedintronIntronof10-20nucleotides1.Endo-nuclease2.Phospho-diesterase3.Kinase(ATP)4.Ligase(ATP)5.Phosphatase+SplicedtRNACCA at 3 end of tRNAsAll tRNAs end in the sequence CCA.Amino acids are added to the CCA end during “charging” of tRNAs for translation.For most eukaryotic tRNAs, the CCA is added after transcription, in a reaction catalyzed by tRNA nucleotidyl transferase.All of the four bases in tRNA can be modified PathologiesresultingfromaberrantsplicingcanbegroupedintwomajorcategoriesMutations affecting proteins that are involved in splicingExamples:Spinal Muscular AtrophyRetinitis PigmentosaMyotonic DystrophyMutations affecting a specific messenger RNA and disturbing its normal splicing patternExamples:-ThalassemiaDuchenne Muscular DystrophyCystic FibrosisFrasier SyndromeFrontotemporal Dementia and ParkinsonismIntron Advantage?One benefit of genes with introns is a phenomenon called alternative splicingA pre-mRNA with multiple introns can be spliced in different waysThis will generate mature mRNAs with different combinations of exonsThis variation in splicing can occur in different cell types or during different stages of developmentIntron Advantage?The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single geneThis allows an organism to carry fewer genes in its genomeDo you believe? RNA interference or RNAi is a remarkable process whereby small noncoding RNA silence specific genes. - RNAi was first observed in plant in immune response to viral pathgens. - MicroRNA regulate gene expression in organisms from nematode to man.RNAInterferenceandInterferenceRNANobel Prize in Physiology or Medicine 2006, Andrew . Fire and Craig . MelloRNAInterference:AMechanismforSilencingGeneExpression1. Small dsRNA fragments can silence the expression of a matching gene. This is RNA interference (RNAi), recently discovered in C. elegans.a. Injecting dsRNA into adult worms results in specific loss of the corresponding mRNA in the worm and its progeny.b. RNAi also occurs in many other organisms, where it protects against viral infection and regulates developmental processes.2. RNAi is highly specific and sensitive, with only a few molecules of dsRNA needed, making it an excellent research tool.Comparison of siRNA and miRNAPrecursorStructureFunctionTargetmRNABiologicalsiRNAEndogenousorexogenousdsRNAdsRNAmRNAcleavageperfectcomplimentarityInhibittransponandvirusinfectionmiRNAEndogenoustranscriptssRNATranslationinhibitionandmRNAcleavageImperfectcomplimentaritydevelopmentForeignDNAandTransgeneForeign DNA and TransgeneAberrant sense RNAdsRNAsiRNAsHeterochromatin formation and Transcriptional silencingRdRPDicerNature2004,Vol431,Sept.16:343Mitrons :Short intronic hairpins pri-miRNApre-miRNAsMicro RNAs (MiRNAs) 22NTsRNA Ploymerase II or IIIDicerSelfsplicingmiRNARISCNo need of DroshaSplicing machinery Lariat debranching enzymeCell 130, July 13 2007: 89-100lncRNA functionsSomething I may not care , but you have to.