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Chapter 14 Opener RNA Splicing (In Eukaryotes) © 2014 Pearson Education, Inc. Figure 14-1 Typical eukaryote gene © 2014 Pearson Education, Inc. Figure 14-2 © 2014 Pearson Education, Inc. Figure 14-3 Sequences within the RNA determine where splicing occurs (Acceptor site) (Donor site) N: any base R: purine Y: pyrimidine © 2014 Pearson Education, Inc. Figure 14-4 Splicing reaction by transesterification © 2014 Pearson Education, Inc. Figure 14-5 © 2014 Pearson Education, Inc. Spliceosome machinery -snRNA: U1, U2, U4, U5, U6 RNA splicing is performed by a large complex called the spliceosome. © 2014 Pearson Education, Inc. Figure 14-6 Some RNA-RNA hybrids formed during splicing reaction: In some cases, different snRNAs recognize the same sequencers in pre-mRNA at different stage of splicing. Branchpoint binding protein (BBP) © 2014 Pearson Education, Inc. Figure 14-7 snRNAs: U1, U2, U4, U5, U6 These RNAs (of 100-300 nucleotides) are complexed with several proteins and called snRNPs (pronounced “snurps”) © 2014 Pearson Education, Inc. (U2 auxiliary factor) © 2014 Pearson Education, Inc. © 2014 Pearson Education, Inc. tri-snRNP particle (U2-U5-U6) © 2014 Pearson Education, Inc. Table 14-1 © 2014 Pearson Education, Inc. Spliceosome assembly (exon definition) is dynamic and variable and its disassembly ensures that the splicing reaction goes only forward in the cell. Assembly can be variable : splicing pairing can occur before or after tri-snRNAP(U4-U5-U5) recruitment Dissembly is driven by one of the DEADbox helicase proteins (called Prp22) © 2014 Pearson Education, Inc. Figure 14-8 © 2014 Pearson Education, Inc. Figure 14-8a © 2014 Pearson Education, Inc. Figure 14-8b © 2014 Pearson Education, Inc. Figure 14-8c © 2014 Pearson Education, Inc. Figure 14-9 Proposed folding of the RNA catalytic regions for splicing of pre-mRNA and group II intron © 2014 Pearson Education, Inc. How does the spliceosome find the splice sites correctly? -One human gene of 363 exons (usually 7 to 8 exons) -Drosophila gene of 38,000 alternative ways of splicing -Average exon is only some 150 nucleotides long -Whereas average intron is about 3000 nucleotides long (Some introns can be as long as 800,000 nucleotides) © 2014 Pearson Education, Inc. Figure 14-10 How does the spliceosome find the splice sites correctly? Errors produced by mistakes in splice site selection © 2014 Pearson Education, Inc. Two ways in which the accuracy of splice-site selection can be enhanced are as follows. One way: When 5’ splice site is encountered in the newly synthesized RNA, the factors that recognize that site(5’ splice site), transferred from C-terminal tail of RNA polymerase II onto RNA. Once in place, the 5’ splice site components are poised to interact with those other factors that binds to the next 3’ splice site to be synthesize. Thus correct 3’ splice site can be recognized before any competing sites further downstream transcribed. © 2014 Pearson Education, Inc. Figure 14-11 Second way by SR to bind to exonic splicing enhancers (ESEs): SR(serine-arginine rich) proteins recruits spliceosome components to the 5’ and 3’ splice sites than to incorrect sites not close to exon. © 2014 Pearson Education, Inc. Figure 14-12 Alternative splicing by SR (trans-splicing) © 2014 Pearson Education, Inc. Figure 14-13 AT-AC(minor) spliseosome catalyzed splicing A small group of introns is spliced by an alternative (minor) spliceosome composed of a different set of snRNPs (perhaps one in 1000 exons in human) © 2014 Pearson Education, Inc. Figure 14-14 Alternative splicing in the troponin T gene At least 40% of Drosophila and as many as 90% of human genes undergoes alternative splicing. © 2014 Pearson Education, Inc. Figure 14-15 Five ways of splice an RNA © 2014 Pearson Education, Inc. Figure 14-16 Alternative splicing of SV40 T antigen Splicing out intron for Large T Splicing out intron for small t © 2014 Pearson Education, Inc. Figure 14-17 Mutually exclusive splicing: Steric(입체적) hindrance(방햬) U1-snRNP U1-snRNP: now U2 snRNP can not bind to branchpoint but bind to U2 snRNP bind first here © 2014 Pearson Education, Inc. Figure 14-18 Mutually exclusive splicing: Combination of major and minor splice sites Non-sense mediated decay © 2014 Pearson Education, Inc. Figure 14-19 Curious Drosophila Dscam (down syndrome cell adhesion molecule) gene: Mutually exclusive splicing on a grand scale (encode 38,016 protein sioforms) © 2014 Pearson Education, Inc. Figure 14-20 Mutually exclusive splicing of Dscam exon 6 can not be accounted for by an standard mechanism and instead use a novel strategy (docking site: select sequences) © 2014 Pearson Education, Inc. Figure 14-21 © 2014 Pearson Education, Inc. Box 14-3-1 © 2014 Pearson Education, Inc. Box 14-3-2 © 2014 Pearson Education, Inc. Figure 14-22 Alternative (대체)splicing(이 어맞추기) is regulated by activators and repressors. © 2014 Pearson Education, Inc. Figure 14-22a Exonic (or intronic) splicing silencers (ESS or ISS): sites that repressor protein binds © 2014 Pearson Education, Inc. Figure 14-22b Exonic (or intronic) splicing enhancers (ESE or ISE): sites that SR protein binds SR protein has RMM (RNA recognition motif domain) and RS domain (rich in arginine and serine) (SR protein) © 2014 Pearson Education, Inc. Figure 14-23 Two mechanisms of silencer action A: A1(repressor) competes off SC35(activator) after binding to ESS. silencer enhancer Heterogeneous nuclear ribonucleoprotein (hnRNP) B: PTB(hnRNP, Pyrimidine binding protein) interact with U1 at the 5’ splicing site and then block U1 to interact with 3’ splicing site. So U1 pairs with exon at downstream. © 2014 Pearson Education, Inc. (repressor) Figure 14-24 Regulation of alternative splicing determines the sex of flies (difference in ratio of activator and repressor determine sex) sis-a and sis-b = Sxl activators is in X chromosome Dpn (Deadpan) = Sxl repressor is in autosome X means X chromosome A means autosome Sxl: sex lethal © 2014 Pearson Education, Inc. Pe: promoter for establishment Pm: promoter for maintenance Figure 14-25 Dsx: double sex gene © 2014 Pearson Education, Inc. Figure 14-26 An alternative splicing switch(대체이어맞추기 전환) lies at the heart(중추) of pluripotency(만능유도) FOXP1 (Transcription factor: Forkhead family of DNA binding protein) FOXP1-ES (protein encode by 17-18b-19 mRNA) FOXP1-ES activates genes OCT4 and NANOG, etc. Induced pluripotent stem cells (iPS cells) © 2014 Pearson Education, Inc. FOXP1 (protein encode by 17-18a-19 mRNA ) Shuffle: mix cards (poker game) Exon shuffling Exon are shuffled by recombination to produce genes encoding new proteins. Intron early model Intron late model: Introns were added later in evolution. There is possibly another advantage afforded these organisms: having coding sequence of genes divided into several exons allows new genes to be created by reshuffing exons. © 2014 Pearson Education, Inc. Figure 14-27 Exons encode protein domains. Borders between exons and introns within a gene coincide with boundaries between domains. © 2014 Pearson Education, Inc. Figure 14-28 Genes made up of parts of other genes © 2014 Pearson Education, Inc. Figure 14-29 Accumulation, loss, and reshuffling of domains during the evolution of a family of proteins © 2014 Pearson Education, Inc. Figure 14-30 RNA editing is another way of altering the sequence of an mRNA Stop codon © 2014 Pearson Education, Inc. Figure 14-31 RNA editing by deaminase Inosine base pair with cytosine. So can alter sequence of protein ADAR (adenosine deaminase acting on RNA) © 2014 Pearson Education, Inc. Figure 14-32 RNA editing by guide RNA-mediated U insertion (in trypanosome coxII gene RNA) © 2014 Pearson Education, Inc. Figure 14-32a © 2014 Pearson Education, Inc. Figure 14-32b © 2014 Pearson Education, Inc. Figure 14-32c © 2014 Pearson Education, Inc. Figure 14-33 Transport of mRNAs (active transport) out of the nucleus through nuclear pore complex © 2014 Pearson Education, Inc.