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1、Ch 12: Mechanisms of transcription Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic code1Part III: Expression of the GenomeCHAPTER 13RNA SplicingMolecular Biology CourseOUTLINE31.The Chemistry of RNA Splicing 2.The Spliceosome Machinery3.Splicing Pathway4.Alternative Splicing5.Exon Shuffling6
2、.RNA Editing7.mRNA TransportMost of the eukaryotic genes are mosaic (嵌合体嵌合体), consisting of intervening sequences separating the coding sequenceExons (外外显子子): the coding sequencesIntrons (内含子内含子) : the intervening sequencesRNA splicing(RNA 剪接）剪接）: the process by which introns are removed from the pr
3、e-mRNA.Alternative splicing (可可变剪接剪接): 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?uHow are the introns and exons distinguished
4、from each other?uHow are exons joined with high precision?uHow 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
5、 human5 splicing site: GU3 splicing site: AGBranch site: A , close 3 and Py tractn5splice 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): 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
6、intron, which is followed by a polypyrimidine tract (Py tract).The intron is removed in a Form Called a Lariat (套套马索索) as the Flanking Exons are joinedTwo successive transesterification（转酯反反应）:Step 1: The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the
7、 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 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
8、3 exons are joined and the intron is liberated in 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 Spl
9、icingExcised intron has a 3-OH groupPhosphorus atom 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.
10、Demonstration of a critical signal within a yeast 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
11、comprises about 150 proteins and 5 snRNAs(small nuclear RNA).Many functions of the spliceosome are carried out by its RNA components.The spliceosome machineryuThe five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nuclear RNAs (snRNAs).uThe complexes of snRNA and proteins are called sma
12、ll nuclear ribonuclear proteins (snRNP, pronounces “snurps”).uThe 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. Catal
13、yzing (or helping to catalyze) the RNA cleavage.RNA-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, he
14、lps other protein, branch-point-binding protein(BBP), 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 11.U1 recognize 5 splice site
15、.2.One subunit of U2AF binds to Py tract and the other to the 3 splice site. The former subunits interacts with branch-point-binding protein (BBP) and helps it bind to the branch point.3.Early (E) complex is formedSplicing pathways Assembly step 21. U2 binds to the branch site, and then A complex is
16、 formed.2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded. This A residue is available to react with the 5 splice site.A被被挤压32Assembly step 31. A rearrangement of the A complex to bring together all three splicing sites. U4, U5 and U6 form the tri-snRN
17、P Particle. 2. With the entry of the tri-snRNP, the A complex 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 1:1.Formation of the
18、C complex: U4 is released from the complex, allowing U6 to interact with U2. This rearrangement, called the c complex, produces the active site.2.Formation of the active site: Juxtaposes (并并置置) the 5 splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5 spl
19、ice site to accomplish the first transesterfication (转酯) reaction.Catalysis 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 rea
20、ction2nd reactionSplicing scheme of adenovirus E1A gene and RNase protection 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
21、 normally does not appear until late in infection.Lane 4, mutant hr440 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
22、apparent. Lane 7, mutant pm1114 plus mutant U1 snRNA (U16a). Even though 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 reliablyS
23、plicing 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. Errors Produced by Mistakes in Splice-site SelectionReasons for the recognition errors(1)The average exon is 150 nt (?), and
24、 the average intron is about 3,000 nt long (some introns are near 800,000 nt). It 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 f
25、or 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 II a
26、re readily recognized, thus preventing exon skipping. 2.There is a mechanism to 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 rec
27、ruiting the splicing machinery to those sites.Two ways to enhance the accuracy of 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
28、the 5 and 3 Splice Sites1.Ensure the accuracy and efficacy of constitutive splicing (组成性剪接成性剪接).2.Regulate alternative splicing.3.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 Esse
29、ntial for SplicingTopic 4 ALTERNATIVE SPLICINGCHAPTER 13 RNA SplicingSingle Genes Can Produce 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.Alt
30、ernative splicingAlternative Splicing in the Troponin T Gene54Different Ways of Alternative 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 E
31、xclusive Splicing互不相容性剪接互不相容性剪接Steric Hindrance: 空空间位阻位阻Combinations of Major and Minor Splice Sites: 主要剪接位点和次要剪接位点的主要剪接位点和次要剪接位点的联合合Nonsense-Mediated Decay: 无无义介介导降解降解Alternative splicingSteric Hindrance: 空空间位阻位阻58Combinations of Major and Minor Splice Sites: 主要剪接位点和次要剪接位点的主要剪接位点和次要剪接位点的联合合59Nonsen
32、se-Mediated Decay: 无无义介介导降解降解60Alternative splicing is regulated by activators and repressorsThe regulating sequences : exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). Alternative splicingnActivators are proteins bind to enhancers to enhance splicing. nRepressors are
33、 proteins bind to silencers to repress splicing.A reporter construct to detect ESS activity62uOne domain is the RNA-recognition motif (RRM), which is responsible for RNA binding. uThe other domain is the RS domain rich in arginine and serine, which mediates interactions between the SR proteins and p
34、roteins within the splicing machinery to promote splicing at the nearby splice sites.SR proteins are splicing activators and contain two domains.1.Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family. 2.These proteins bind RNA, but lack the RS domains. Therefore,
35、(1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.hnRNPs are Splicing Repressors Regulated Alternative SplicingBinds at each end of the exon and conceals (隐藏藏) it Coats the RNA and makes the exons invisible to the splicing machineryTwo
36、 models for the action of a repressor hnRNPI/PTB in inhibiting splicing1.Producing multiple protein products, called isoforms. They can have similar, distinct or antagonistic functions. One gene encodes multiple functions2.Switching on and off the expression of a given gene that encodes only one fun
37、ction. When the exon containing a stop is included to produce nonfunctional protein, or the intron is included to prevent mRNA transport The outcome of alternative splicing (可变剪接的结果/生物学功能)Topic 5 Exon Shuffling外外显子改子改组CHAPTER 13 RNA SplicingWhere is the introns from?Model 1: Intron Early ModelModel
38、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 between two nonallelic genes are occasionally mixed. Gilbert suggested that exons might each encode a single protein domain, establishing a kind of
39、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 from different combinations of such exons and their resulting domain combinations.The process of exon shuffling can create a mosaic, or chimeric
40、, 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 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 co
41、ntext of evolution, exon shuffling is significant due to its ability to quickly create new multidomain proteins , leading to increased variance in species.70Exons Encode Protein Domain71Gene Made up of Parts of Other Genes7273Topic 6RNA EDITINGCHAPTER 13 RNA SplicingRNA editing is another way of cha
42、nging the sequence of an mRNA at the RNA level I. Site specific deamination (位点特异性去位点特异性去氨反氨反应):1. A specifically targeted C residue within mRNA is converted into U by the deaminase (脱氨脱氨酶酶). The process occurs only in certain tissues or cell types and in a regulated manner.RNA editingFigure 13-25St
43、op codeIn liverIn intestinesFigure 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 prot
44、ein. II Guide RNA-directed uridine insertion or deletion.1. This form of RNA editing is found in the mitochondria of trypanosomes（锥虫）虫）.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
45、, completely altering the “meaning” of the message.4. Us are inserted into the message by guide RNAs (gRNAs) .Having three regions: anchor directing the gRNAs to the region of mRNAs it will edit. editing region determining where the Us will be inserted poly-U stretch gRNAsFigure 13-26 RNA editing by
46、 gRNA-mediated U insertionTopic 7mRNA TRANSPORTCHAPTER 13 RNA SplicingOnce processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translationAll the fully processed mRNAs are transported to the cytoplasm for translation into proteinsmRNA transportFigure 13-271.Why RNA splic
47、ing is important? 2.Chemical reaction: determination of the splice sites, the products, trans-splicing3.Spliceosome: splicing pathway and finding the splice sites.4.Self-splicing introns and mechanisms5.Alternative splicing and regulation, alternative spliceosome6.Two different mechanisms of RNA editing7.mRNA transport-a link to translation Key points of the chapter
