分子生物学英文版教学课件：Ch13 RNA Splicing

1、Welcome Each of You to My Molecular Biology ClassMolecular Biology of the Gene, 5/E - Watson et al. (2004)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods4/3/05Ch 12 : Mechanisms of TranscriptionCh 13 : RNA Splicin

2、gCh 14 : TranslationCh 15 : The Genetic code4/3/05Molecular Biology CourseFigure 13-1Primary transcriptMost of the eukaryotic genes are mosaic (嵌合体嵌合体), consisting of intervening sequences separating the coding sequencenExons (外显子外显子): the coding sequencesnIntrons (内含子内含子) : the intervening sequence

3、snRNA splicing: the process by which introns are removed from the pre-mRNA.nAlternative splicing (可变剪接可变剪接): some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 60% of the human genes are spliced in this manner.CHAPTER 13 RNA SplicingSequences within the RNA Determine

4、Where Splicing OccursThe chemistry of RNA splicingThe borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.Figure 13-2The consensus sequences for humann5splice site (5剪接位点剪接位点): the exon-intron boundary at the 5 end of the intronn3 splice site (3剪接位点剪接位点

5、): the exon-intron boundary at the 3 end of the intronnBranch point site (分枝位点分枝位点): an A close to the 3 end of the intron, which is followed by a polypyrimidine tract (Py tract).The intron is removed in a Form Called a Lariat (套马索套马索) as the Flanking Exons are joinedTwo successive transesterificati

6、on:Step 1: The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5 splice site. As a result, the 5 exon is released and the 5-end of the intron forms a three-way junction structure.The chemistry of RNA splicingFigure 13-3Three-way junction The structure

7、of three-way junctionFigure 13-4This figure has an errorIntron5 endStep 2: The OH of the 5 exon attacks the phosphoryl group at the 3 splice site. As a consequence, the 5 and 3 exons are joined and the intron is liberated in the shape of a lariat.Figure 13-3Exons from different RNA molecules can be

8、fused by Trans-splicingnTrans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. The chemistry of RNA splicingTrans-splicingFigure 13-5Not a lariatCHAPTER 13 RNA SplicingRNA splicing is carried out by a large complex called spliceosomenThe above desc

9、ribed splicing of introns from pre-mRNA are mediated by the spliceosome.nThe spliceosome comprises about 150 proteins and 5 snRNAs.nMany functions of the spliceosome are carried out by its RNA components.The spliceosome machinerynThe five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nu

10、clear RNAs (snRNAs).nThe complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP, pronounces “snurps”).nThe spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reactionnThree roles of snRNPs in splicing1. Recognizing the 5 s

11、plice site and the branch site.2. Bringing those sites together.3. Catalyzing (or helping to catalyze) the RNA cleavage.RNA-RNA, RNA-protein and protein-protein interactions are all important during splicing.Figure 13-6RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNACHA

12、PTER 13 RNA SplicingAssembly, rearrangement, and catalysis within the spliceosome: the splicing pathway (Fig. 13-8)Assembly step 11.U1 recognize 5 splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3 splice site. The former subunits interacts with BBP and helps it bind to the

13、 branch point.3. Early (E) complex is formedSplicing pathways Assembly step 21.U2 binds to the branch site, and then A complex is formed.2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded (Figure 13-6). This A residue is available to react with the 5 sp

14、lice site.Figure 13-8E complexA complexFigure 13-6bnAssembly step 31. U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.Figure 13-8A complexB complexnAssembly step 4U1 leaves the complex, and U6 replaces it at the 5 splice si

15、te.U4 is released from the complex, allowing U6 to interact with U2 (Figure 13-6c).This arrangement called the C complex.Figure 13-8Figure 13-6cB complexC complex in which the catalysis has not occurred yetCatalysis Step 1:Formation of the produces the active site, with U2 and U6 RNAs being brought

16、togetherFormation of the juxtaposes (并置并置) the 5 splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5 splice site to accomplish the first transesterfication (转酯转酯) reaction.Catalysis Step 2: helps to bring the two exons together, and aids the second transe

17、sterification reaction, in which the 3-OH of the 5 exon attacks the 3 splice site.nFinal Step: Release of the mRNA product and the snRNPsFigure 13-8C complex 1st reaction2nd reactionE complexA complexB complexC complex （没有（没有该该complex的图）的图）splicesome-mediated splicing reactionsFigure 13-8How does sp

18、liceosome find the splice sites reliablySplicing pathways Two kinds of splice-site recognition errorsnSplice sites can be skipped.n“Pseudo” splice sites could be mistakenly recognized, particularly the 3 splice site. Figure 13-12Reasons for the recognition errors(1) The average exon is 150 nt (?), a

19、nd the average intron is about 3,000 nt long (some introns are near 800,000 nt)nIt is quite challenging for the spliceosome to identify the exons within a vast ocean of the intronic sequences. (2) The splice site consensus sequence are rather loose. For example, only AG G tri-nucleotides is required

20、 for the 3 splice site, and this consensus sequence occurs every 64 nt theoretically. 1. Because the C-terminal tail of the RNA polymerase II carries various splicing proteins, co-transcriptional loading of these proteins to the newly synthesized RNA ensures all the splice sites emerging from RNAP I

21、I are readily recognized, thus preventing exon skipping. Two ways to enhance the accuracy of the splice-site selection2. There is a mechanism to ensure that the splice sites close to exons are recognized preferentially. SR proteins bind to the ESEs (exonic splicing enhancers) present in the exons an

22、d promote the use of the nearby splice sites by recruiting the splicing machinery to those sitesSR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites. Figure 13-131.Ensure the accuracy and efficacy of consti

23、tutive splicing (组成性剪接组成性剪接).2.Regulate alternative splicing3.There are many varieties of SR proteins. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns. SR proteins are essential for splicingCHAPTER 13 RNA SplicingMany genes in higher eukary

24、otes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products.Single genes can produce multiple products by alternative splicingAlternative splicingDrosophila DSCAM gene can be spliced in 38,000 alternative ways Figure 13-13Fig

25、ure 13-15Different ways of alternative splicingFigure 13-14Alternative splicing can be either constitutive or regulatednConstitutive alternative splicing: more than one product is always made from a pre-mRNAnRegulative alternative splicing: different forms of mRNA are produced at different time, und

26、er different conditions, or in different cell or tissue typesAn example of constitutive alternative splicing : Splicing of the SV40 T antigen RNAFigure 13-16Alternative splicing is regulated by activators and repressorsAlternative splicingn The regulating sequences : exonic (or intronic) splicing en

27、hancers (ESE or ISE) or silencers (ESS and ISS). nActivators are proteins bind to enhancers to enhance splicing. nRepressors are proteins bind to silencers to repress splicing.SR proteins are splicing activators and contain two domains.(1)One domain is the RNA-recognition motif (RRM), which is respo

28、nsible for RNA binding. (2)The other domain is the RS domain rich in arginine and serine, which mediates interactions between the SR proteins and proteins within the splicing machinery to promote splicing at the nearby splice sites.hnRNPs are splicing repressors 1.Most silencers are recognized by hn

29、RNP ( heterogeneous nuclear ribonucleoprotein) family. 2.These proteins bind RNA, but lack the RS domains. Therefore, (1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.Regulated alternative splicingFigure 13-17Binds at each end of the

30、exon and conceals (隐藏隐藏) it Coats the RNA and makes the exons invisible to the splicing machineryTwo models for the action of a repressor hnRNPI/PTB in inhibiting splicingFigure 13-18The outcome of alternative splicing (可变剪接的结果可变剪接的结果/生物学功能生物学功能)1.Producing multiple protein products, called isoforms

31、. They can have similar, distinct or antagonistic functions. One gene encodes multiple functions 2. Switching on and off the expression of a given gene that encodes only one function. When the exon containing a stop is included to produce nonfunctional protein, or the intron is included to prevent m

32、RNA transport A small group of intron are spliced by minor spliceosomenIt splices introns harboring determinant sequences distinct from those recognized by the major spliceosome. nIt is known AT-AC spliceosome. The termini of the originally identified introns that is splice contain AU at 5ss, and AC

33、 at the 3 ss. nThe chemical pathway is the same as the major spliceosome, but U11 and U12 are used in places of U1 and U2, respectively.Alternative splicingFigure 13-19 The AT-AC spliceosomeFigure 13-20 Sequences conserved in different kinds of introns.Trans-splicing自剪接内含子自剪接内含子CHAPTER 13 RNA Splici

34、ngSelf-splicing introns reveal that RNA can catalyze splicingSelf-splicing introns: -Introns that can fold into a specific conformation within the precursor RNA, and catalyze the chemistry of their own release and the exon ligation. -They can remove themselves from pre-RNAs in the absence of any pro

35、teins or other RNAs in vitro. Splicing pathways fold into a specific conformationcatalyze the chemical reaction using metal ions as cofactorsThere are two classes of self-splicing introns:ngroup I self-splicing intronsngroup II self-splicing introns.TABLE 13-1 Three classes of RNA SplicingClassAbund

36、anceMechanismCatalytic MachineryNuclear pre-mRNAVery common; most eukaryotic genesTwo sequential transesterification reactions; branch site AspliceosomeGroup II intronsRare; some eukaryotic genes from organelles and prokaryotesSame as pre-mRNARNA enzyme encoded by intron (ribozyme)Group I intronsRar

37、e; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genesTwo sequentialtransesterification reactions; exogenous GSame as group II intronsFigure 13-9The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.The similar

38、ity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterificationFigure 13-10Group I introns release a linear intron rather than a lariatnDuring the 1st transesterification reaction, group I introns use a free G, instead of using a branch point A, to attac

39、k the 5 splice site.nAs a result, this G is attached to the 5 end of the intron.nA 3-OH group is resulted at the 5 exon, which then attacks the 5 splice site for the 2nd transesterification reaction. This is the same as that of splicing of the group II and pre-mRNA introns. Splicing pathways G inste

40、ad of Aa linear introna Lariat intronFigure 13-91.Smaller than group II introns2.Share a conserved secondary structure, which includes an “internal guide sequence” base-pairing with the 5 splice site sequence in the upstream exon.3.Their tertiary structure contains a binding pocket that will accommo

41、date the guanine nucleotide or nucleoside cofactor.Group I introns Adams et al., Nature 2004, Crystal structure of a self-splicing group I intron with both exons. Group I intron website: 2D structure3D structureCHAPTER 13 RNA SplicingRNA editing is another way of changing the sequence of an mRNA at

42、the RNA level I. Site specific deamination (位位点特异性去氨反应点特异性去氨反应):1. A specifically targeted C residue within mRNA is converted into U by the deaminase (脱氨酶脱氨酶). The process occurs only in certain tissues or cell types and in a regulated manner.RNA editingFigure 13-25Stop codeIn liverIn intestinesFigu

43、re 13-25 RNA editing by deamination. The human apolipoprotein gene2. Adenosine deamination also occurs in cells. The enzyme ADAR (adenosine deaminase acting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alter the sequence of the protein. II Guide RNA-directed uridi

44、ne insertion or deletion.1. This form of RNA editing is found in the mitochondria of trypanosomes.2. Multiple Us are inserted into specific region of mRNAs after transcription (or US may be deleted).3. The addition of Us to mRNA changes codons and reading frames, completely altering the “meaning” of the message.4. Us are inserted into the m