1、Ch 12: Mechanisms of transcription Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic code1Part III: Expression of the GenomeCHAPTER 13RNA SplicingMolecular Biology CourseOUTLINE3The Chemistry of RNA Splicing The Spliceosome MachinerySplicing PathwayAlternative SplicingExon ShufflingRNA Editing

2、mRNA TransportMost of the eukaryotic genes are mosaic (嵌合體), consisting of intervening sequences separating the coding sequenceExons (外顯子): the coding sequencesIntrons (內(nèi)含子) : the intervening sequencesRNA splicing(RNA 剪接）: the process by which introns are removed from the pre-mRNA.Alternative splici

3、ng (可變剪接): some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 90% of the human genes are spliced in this manner.Figure 13-18Topic 1 : THE CHEMISTRY OF RNA SPLICINGCHAPTER 13 RNA SplicingQuestion?How are the introns and exons distinguished from each other?How are exons

4、 joined with high precision?How are introns removed?10Sequences within the RNA Determine Where Splicing OccursThe borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.The chemistry of RNA splicingThe consensus sequences for human5 splicing site: GU3 spli

5、cing site: AGBranch site: A , close 3 and Py tract5splice site (5剪接位點(diǎn)): the exon-intron boundary at the 5 end of the intron3 splice site (3剪接位點(diǎn)): the exon-intron boundary at the 3 end of the intronBranch point site (分枝位點(diǎn)): an A close to the 3 end of the intron, which is followed by a polypyrimidine

6、tract (Py tract).The intron is removed in a Form Called a Lariat (套馬索) as the Flanking Exons are joinedTwo successive transesterification（轉(zhuǎn)酯反應(yīng)）: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 releas

7、ed and the 5-end of the intron forms a three-way junction structure.The chemistry of RNA splicingThe structure of three-way junctionIntron5 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

8、 the shape of a lariat.Exons from different RNA molecules can be fused by Trans-splicingTrans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. The chemistry of RNA splicingSimplified Mechanism of SplicingExcised intron has a 3-OH groupPhosphorus at

9、om between 2 exons in spliced product comes from 3-splice siteIntermediate and spliced intron contain a branched nucleotide Branch involves 5-end of intron binding to a site within the intronTime course of intermediate and liberated intron appearance.Demonstration of a critical signal within a yeast

10、 intron21Topic 2 THE SPLICESOME MACHINERYCHAPTER 13 RNA SplicingYeast spliceosomes23RNA splicing is carried out by a large complex called spliceosomeThe above described splicing of introns from pre-mRNA are mediated by the spliceosome.The spliceosome comprises about 150 proteins and 5 snRNAs(small n

11、uclear RNA).Many functions of the spliceosome are carried out by its RNA components.The spliceosome machineryThe five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nuclear RNAs (snRNAs).The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP, pronounces

12、“snurps”).The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reactionThree roles of snRNPs in splicing1. Recognizing the 5 splice site and the branch site.2. Bringing those sites together.3. Catalyzing (or helping to catalyze) the RNA cleavage.RNA-

13、RNA, RNA-protein and protein-protein interactions are all important during splicing.RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNANon-snRNPU2AF:1.recognize the Py tract and 3 splicing site 2 . In the initial step, helps other protein, branch-point-binding protein(BBP)

14、, bind to the branch site.RNA-annealing factorsDEAD-box helicase protein28Topic 3 SPLICING PATHWAYSCHAPTER 13 RNA SplicingAssembly, rearrangement, and catalysis within the spliceosome: the splicing pathwayAssembly step 1U1 recognize 5 splice site.One subunit of U2AF binds to Py tract and the other t

15、o the 3 splice site. The former subunits interacts with branch-point-binding protein (BBP) and helps it bind to the branch point.Early (E) complex is formedSplicing pathways Assembly step 2 U2 binds to the branch site, and then A complex is formed.2. The base-pairing between the U2 and the branch si

16、te is such that the branch site A is extruded. This A residue is available to react with the 5 splice site.A被擠壓32Assembly step 31. A rearrangement of the A complex to bring together all three splicing sites. U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex

17、 is converted into the B complex.Assembly step 41. U1 leaves the complex, and U6 replaces it at the 5 splice site.2. Those steps complete the assembly pathway. The next rearrangements triggers catalysis.Catalysis Step 1:Formation of the C complex: U4 is released from the complex, allowing U6 to inte

18、ract with U2. This rearrangement, called the c complex, produces the active site.Formation of the active site: Juxtaposes (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 (轉(zhuǎn)酯) reaction.C

19、atalysis Step 2:U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3-OH of the 5 exon attacks the 3 splice site.Final Step: Release of the mRNA product and the snRNPs1st reaction2nd reactionSplicing scheme of adenovirus E1A gene and RNase p

20、rotection assay to detect each spliced product.Lane 1, size markersLane 2, mock-transfected cellsLane 3, wild-type E1A gene with wild-type U1 snRNA. Signals were visible for the 13S and 12S products, but not for the 9S product, which normally does not appear until late in infection.Lane 4, mutant hr

21、440 with an altered 12S 5-splice site. No 12S signal was apparent. Lane 5, mutant hr440 plus mutant U1 snRNA (U14u). Splicing at the 12S 5-site was restored. Lane 6, mutant pm1114 with an altered 13S 5-splice site. No 13S signal was apparent. Lane 7, mutant pm1114 plus mutant U1 snRNA (U16a). Even t

22、hough base pairing between the 5-splice site and U1 snRNA was restored, no 13S splicing occurred.The spliceosome cycle42Three Classes of RNA Splicing4344Group I and Group II IntronsHow does spliceosome find the splice sites reliablySplicing pathways Two kinds of splice-site recognition errorsSplice

23、sites can be skipped.“Pseudo” splice sites could be mistakenly recognized, particularly the 3 splice site. Errors Produced by Mistakes in Splice-site SelectionReasons for the recognition errorsThe average exon is 150 nt (?), and the average intron is about 3,000 nt long (some introns are near 800,00

24、0 nt). It is quite challenging for the spliceosome to identify the exons within a vast ocean of the intronic sequences. The splice site consensus sequence are rather loose. For example, only AGG tri-nucleotides is required for the 3 splice site, and this consensus sequence occurs every 64 nt theoret

25、ically. 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 II are readily recognized, thus preventing exon skipping. There is a mechanism to

26、ensure that the splice sites close to exons are recognized preferentially. Serine-rich (SR) proteins bind to the ESEs (exonic splicing enhancers) present in the exons and promote the use of the nearby splice sites by recruiting the splicing machinery to those sites.Two ways to enhance the accuracy o

27、f the splice-site selectionSR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites. SR Protein Recruit Spliceosome Components to the 5 and 3 Splice SitesEnsure the accuracy and efficacy of constitutive splicin

28、g (組成性剪接).Regulate alternative splicing.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 SplicingTopic 4 ALTERNATIVE SPLICINGCHAPTER 13 RNA SplicingSingle Genes Can Prod

29、uce Multiple Products by Alternative SplicingMany genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products.Alternative splicingAlternative Splicing in the Troponin T Gene54Different Ways of Alternativ

30、e SplicingAn example of constitutive alternative splicing : Splicing of the SV40 T antigen RNAt-ag blocks apoptosis T-ag induces transformation and cell cycle reentrySeveral Mechanism Exits to Ensure Mutually Exclusive Splicing互不相容性剪接Steric Hindrance: 空間位阻Combinations of Major and Minor Splice Sites

31、: 主要剪接位點(diǎn)和次要剪接位點(diǎn)的聯(lián)合Nonsense-Mediated Decay: 無義介導(dǎo)降解Alternative splicingSteric Hindrance: 空間位阻58Combinations of Major and Minor Splice Sites: 主要剪接位點(diǎn)和次要剪接位點(diǎn)的聯(lián)合59Nonsense-Mediated Decay: 無義介導(dǎo)降解60Alternative splicing is regulated by activators and repressorsThe regulating sequences : exonic (or intronic)

32、splicing enhancers (ESE or ISE) or silencers (ESS and ISS). Alternative splicingActivators are proteins bind to enhancers to enhance splicing. Repressors are proteins bind to silencers to repress splicing.A reporter construct to detect ESS activity62One domain is the RNA-recognition motif (RRM), whi

33、ch is responsible for RNA binding. 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.SR proteins are splicing activators and contain two domains.

34、Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family. 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.hnRNPs are Splicing Repressors Reg

35、ulated Alternative SplicingBinds at each end of the exon and conceals (隱藏) it Coats the RNA and makes the exons invisible to the splicing machineryTwo models for the action of a repressor hnRNPI/PTB in inhibiting splicingProducing multiple protein products, called isoforms. They can have similar, di

36、stinct or antagonistic functions. One gene encodes multiple functionsSwitching 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 mRNA transport The outcome of al

37、ternative splicing (可變剪接的結(jié)果/生物學(xué)功能)Topic 5 Exon Shuffling外顯子改組CHAPTER 13 RNA SplicingWhere is the introns from?Model 1: Intron Early ModelModel 2: Intron Late Model 69Exon shufflingExon shuffling is a theory, introduced by Walter Gilbert in 1977, in which different exons either within a gene or betwe

38、en two nonallelic genes are occasionally mixed. Gilbert suggested that exons might each encode a single protein domain, establishing a kind of modular property. In this fashion, it would be possible for exons to essentially be “mixed and matched” to produce a variety of different proteins, yielded f

39、rom different combinations of such exons and their resulting domain combinations.The process of exon shuffling can create a mosaic, or chimeric, protein that is partly built of domains or segments which are similar or identical to domains of other proteins. The mosaic protein is created when an exon

40、 from one gene is integrated into another genes intron. Another kind of exon shuffling is when an exon is duplicated in the same gene.In the context of evolution, exon shuffling is significant due to its ability to quickly create new multidomain proteins , leading to increased variance in species.70

41、Exons Encode Protein Domain71Gene Made up of Parts of Other Genes7273Topic 6RNA EDITINGCHAPTER 13 RNA SplicingRNA editing is another way of changing the sequence of an mRNA at the RNA level I. Site specific deamination (位點(diǎn)特異性去氨反應(yīng)):1. A specifically targeted C residue within mRNA is converted into U by the deaminase (脫氨酶). The process occurs only in certain tissues or cell types and in a regulated manner.RNA editingFigure 13-25Stop codeIn liverIn intestinesFigure 13-25 RNA editing by deamination. The human apolipoprotein gene2. Adenosine deamination also occurs in cells. The enzym