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1、李 希分子医学教育部重点实验室,Transcription and Post-transcription Modification,Post-transcriptional Processing of RNA,Making ends of RNARNA splicing,Primary Transcript,Primary Transcript-the initial molecule of RNA produced-hnRNA（heterogenous nuclear RNA）In prokaryotes,DNA RNA protein in cytoplasm concurrently I
2、n eukaryotes nuclear RNA Cp RNA,Processing of eukaryotic pre-mRNA,Human dystrophin gene has 79 exons,spans over 2,300-Kb and requires over 16 hours to be transcribed!,For primary transcripts containing multiple exons and introns,splicing occurs before transcription of the gene is complete-co-transcr
3、iptional splicing.,Types of RNA processing,A)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 mech
4、anisms.,The RNA Pol II CTD is required for the coupling of transcription with mRNA capping,polyadenylation and splicing,The coupling allows the processing factors to present at high local concentrations when splice sites and poly(A)signals are transcribed by Pol II,enhancing the rate and specificity
5、 of RNA processing.The association of splicing factors with phosphorylated CTD also stimulates Pol II elongation.Thus,a pre-mRNA is not synthesized unless the machinery for processing it is properly positioned.,Time course of RNA processing,5 and 3 ends of eukaryotic mRNA,Add a GMPMethylate it and1s
6、t few nucleotides,Cut the pre-mRNAand add As,5-UTR,3-UTR,Capping of pre-mRNAs,Cap=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 i
7、s only 25-30 bp longCap structure is recognized by CBC（cap-binding complex）stablize the transcript prevent degradation by exonucleases stimulate splicing and processing,Sometimesmethylated,Sometimesmethylated,The cap is added after the nascent RNA molecules produced by RNA polymerase II reach a leng
8、th of 25-30 nucleotides.Guanylyltransferase is recruited and activated through binding to the Ser5-phosphorylated Pol II CTD.The methyl groups are derived from S-adenosylmethionine.Capping helps stabilize mRNA and enhances translation,splicing and export into the cytoplasm.,Capping of the 5 end of n
9、ascent RNA transcripts with m7G,Existing in a single complex,Consensus sequence for 3 process,AAUAAA:CstF(cleavage stimulation factor F)GU-rich sequence:CPSF(cleavage and polyadenylation specificity factor),Polyadenylation of mRNA at the 3 end,CPSF:cleavage and polyadenylation specificity factor.CSt
10、F: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
11、 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 polyA,Need 5 ca
12、p 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 pathw
13、ay 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 splicing,Back
14、ground: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.Sur
15、prise:The RNA is generated from 4 different regions of the DNA!How can weexplain this?Splicing!,The discovery of split genes(1977)1993 Noble Prize in Medicine To Dr.Richard Robert and Dr.Phillip Sharp,The matured mRNAs are much shorter than the DNA templates.,DNA,mRNA,Exon and Intron,Exon is any seg
16、ment 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.,Exons aresimilar in size,Introns are highlyvariable in size,GT-AG rule,GT-AG
17、rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes.Splice sites are the sequences immediately surrounding the exon-intron boundaries Splicing junctions are recognized only in the correct pairwise combinations,The sequence o
18、f 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 characterisit
19、ic 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-tRNA,Group I and II introns,The sequence of transesterification reactio
20、ns that splice together the exons of eukaryotic pre-mRNAs.,Splicing of Group I and II introns,Introns 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-splic
21、ingInitiate splicing with an internal AUses a phosphoester transfer mechanismDoes not require ATP hydrolysis,Self-splicing in pre-rRNA in Tetrahymena:T.Cech et al.1981,Additional proteinsare NOT needed forsplicing of this pre-rRNA!,Do need a G nucleotide(GMP,GDP,GTP or Guanosine).,The sequence of re
22、actions in the self-splicing of Tetrahymena group I intron.,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
23、 the reaction but is not required for catalysis.Thus RNA can be an enzyme.,Enzymes composed of RNA are called ribozymes.,Hammerhead ribozymes,A 58 nt structure is used in self-cleavageThe sequence CUGA adjacent to stem-loops is sufficient for cleavage,Mechanism of hammerhead ribozyme,The folded RNA
24、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,Distribution of Group
25、I introns,Prokaryotes 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 structure,Splicing of pre-mRNA,T
26、he 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 phosph
27、oester transfer mechanism for splicing,Initiation of phosphoester transfers in pre-mRNA,Uses 2 OH of an A internal to the intronForms a branch point 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 lar
28、iatA debranching enzyme cleaves the lariat at the branch to generate a linear intronLinear intron is degraded,Involvement of snRNAs and snRNPs,snRNAs=small nuclear RNAssnRNPs=small nuclear ribonucleoproteins particles(snRNA complex with protein)Addition of these antibodies to an in vitro pre-mRNA sp
29、licing reaction blocked splicing.Thus the snRNPs were implicated in splicing,Recognizing the 5 splice site and the branch site.Bringing those sites together.Catalyzing(or helping to catalyze)the RNA cleavage.,Role of snRNPs in RNA splicing,RNA-RNA,RNA-protein and protein-protein interactions are all
30、 important during splicing,snRNPs,U1,U2,U4/U6,and U5 snRNPsHave snRNA in each:U1,U2,U4/U6,U5Conserved from yeast to humanAssemble into spliceosomeCatalyze splicing,Splicing of pre-mRNA occurs in a“spliceosome”an RNA-protein complex,spliceosome,(100 proteins+5 small RNAs),The spliceosome is a large p
31、rotein-RNA complex in which splicing of pre-mRNAs occurs.,Assembly of spliceosome,snRNPs 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 d
32、issociatesU4U5U6 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 reaction,Steps of the spliceosome-mediated splicing reaction,A schematic diagr
33、am of six rearrangements that the spliceosome undergoes in mediating the first transesterification reaction in pre-mRNA splicing.,Assembly of spliceosome,The spliceosome cycle,The Significance of Gene Splicing,The introns are rare in prokaryotic structural genesThe introns are uncommon in lower euka
34、ryote(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 selection,Mechanisms prev
35、ent splicing error,Co-transcriptional loading processSR proteins recruit spliceosome components to the 5 and 3 splice sites,SR protein=Serine Arginine rich proteinESE=exonic splicing enhancersSR protein regulates alternative splicing,Alternative splicing,Alternative splicing occurs in all metazoa an
36、d is especially prevalent in vertebrate,Five ways to splice an RNA,Regulated alternative splicing,Different signals in the pre-mRNA and different proteins cause spliceosomes to form in particular positions to give alternative splicing,APOPTOSIS,Alternative splicing can generate mRNAs encoding protei
37、ns with different,even opposite functions,(programmedcell death),(+),(-),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.,Drosophila Dscam gene contains thousa
38、nds of possible splice variants,Cis-and Trans-Splicing,Cis-: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-mRNA splicing,trans-mRNA splicing,spliced
39、 leader,Same splicing mechanism is employed in trans-splicing,Spliced leader contains the cap structure!,RNA editing,RNA 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 af
40、ter transcription.Examples:mitochondrial genes in Trypanosomes（锥虫）Can add,delete or change nucleotides by editing,Two mechanisms mediate editing,Guide RNA-directed uridine insertion or deletionSite-specific deamination,Insertion and deletion of nucleotides by editing,Uses a guide RNA(in 20S RNP=edit
41、osome)that is encoded elsewhere in the genomePart of the guide RNA is complementary to the mRNA in vicinity of editing,Trypanosomal RNA editing pathways.(a)Insertion.(b)Deletion.,Mammalian example of editing,The C is converted to U in intestine by a specific deaminating enzyme,not by a guide RNA.,Cu
42、tting and Trimming RNA,Can 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 RNA,The posttranscriptional processing of E.col
43、i rRNA.,RNase III cuts in stems of stem-loops,16S rRNA,23S rRNA,RNase III,No apparent primary sequence specificity-perhaps RNase III recognizes a particular stem structure.,Eukaryotic rRNA Processing,The primary rRNA transcript(7500nt,45S RNA)contains 18S,5.8S and 28SMethylationoccur mostly in rRNA
44、sequence80%:O2-methylribose,20%:bases(A or G)peudouridine 95 U in rRNA in human are converted to Ysmay contribute rRNA tertiary stability,Transfer RNA Processing,Cloverleaf structureCCA:amino acid binding siteAnticodon60 tRNA genes in E.coli,A schematic diagram of the tRNA cloverleaf secondary struc
45、ture.,Endo-and exonucleases to generate ends of tRNA,Endonuclease RNase P cleaves to generate the 5 end.Endonuclease RNase F cleaves 3 nucleotides past the mature 3 end.Exonuclease RNase D trims 3 to 5,leaving the mature 3 end.,Splicing of pre-tRNA,Introns in pre-tRNA are very short(about 10-20 nucl
46、eotides)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 pho
47、sphate on the 2OH(remaining after phosphodiesterase),Steps in splicing of pre-tRNA,P,OH 5,2,3 cyclic phosphate,Excised intron,Intron of 10-20 nucleotides,1.Endo-nuclease,2.Phospho-diesterase3.Kinase(ATP)4.Ligase(ATP)5.Phosphatase,+,+,Spliced tRNA,CCA at 3 end of tRNAs,All tRNAs end in the sequence C
48、CA.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,Pathologies resulting from aberrant splicing can be g
49、rouped in two major categories,Mutations 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
50、 FibrosisFrasier SyndromeFrontotemporal Dementia and Parkinsonism,Intron 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 v
