Chapter 14 Opener RNA Splicing (In Eukaryotes)

Typical eukaryote gene Figure 14-1 Typical eukaryote gene Figure 14-1 Typical eukaryote gene 2

Figure 14-2 Figure 14-2 3

Sequences within the RNA determine where splicing occurs Figure 14-3 Sequences within the RNA determine where splicing occurs (Donor site) (Acceptor site) Figure 14-3 Sequences within the RNA determine where splicing occurs N: any base R: purine Y:pyrimidine Donor site Acceptor site N: any base R: purine Y: pyrimidine 4

by transesterification Figure 14-4 Splicing reaction by transesterification Figure 14-4 Splicing reaction 5

Figure 14-5 Figure 14-5 6

Spliceosome machinery -snRNA: U1, U2, U4, U5, U6 RNA splicing is performed by a large complex called the spliceosome.

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) Figure 14-6 Some RNA-RNA hybrids formed during splicing reaction Spliceosome machinery Branchpoint binding protein (BBP) 8

These RNAs (of 100-300 nucleotides) are 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”) Figure 14-7 9

(U2 auxiliary factor) U2 auxiliary factor

tri-snRNP particle (U2-U5-U6)

Table 14-1 Table 14-1 13

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 DEAD-box helicase proteins (called Prp22) Spliceosome assembly is dynamic and variable and its disassembly ensures that the splicing reaction goes only forward in the cell

Figure 14-8 Figure 14-8 ©1986 Elsevier http://www.sciencedirect.com/science/article/pii/0092867486907518 15

Figure 14-8a Figure 14-8a ©1986 Elsevier http://www.sciencedirect.com/science/article/pii/0092867486907518 16

Figure 14-8b Figure 14-8b ©1986 Elsevier http://www.sciencedirect.com/science/article/pii/0092867486907518 17

Figure 14-8c Figure 14-8c ©1986 Elsevier http://www.sciencedirect.com/science/article/pii/0092867486907518 18

Figure 14-9 Proposed folding of the RNA catalytic regions for splicing of pre-mRNA and group II intron Figure 14-9 Proposed folding of the RNA catalytic regions for splicing of pre-mRNA and group II intron 19

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)

How does the spliceosome find the splice sites correctly? Figure 14-10 How does the spliceosome find the splice sites correctly? Errors produced by mistakes in splice site selection Figure 14-10 How does the spliceosome find the splice sites correctly? Errors produced by mistakes in splice site selection 21

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. Two ways in which the accuracy of splice-site selection can be enhanced are as follows. When 5’ splice site is encountered in the newly synthesized RNA, the factor that recognize that size 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. One way:

Second way by SR to bind to exonic splicing enhancers (ESEs): 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. Figure 14-11 Second way: SR(serine-arginine rich) proteins recruits spliceosome components to the 5’ and 3’ splice sites. 23

Alternative splicing by SR (trans-splicing) Figure 14-12 Alternative splicing by SR (trans-splicing) Figure 14-12 Alternative splicing by SR (trans-splicing) 24

AT-AC(minor) spliseosome catalyzed splicing 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) Figure 14-13 AT-AC(minor) spliseosome catalyzed splicing 25

Alternative splicing in the troponin T gene Figure 14-14 Alternative splicing in the troponin T gene 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. At least 40% of Drosophila and as many as 90% of human genes undergoes alternative splicing. 26

Five ways of splice an RNA Figure 14-15 Five ways of splice an RNA Figure 14-15 27

Alternative splicing of SV40 T antigen Figure 14-16 Alternative splicing of SV40 T antigen Splicing out intron for Large T Splicing out intron for small t Figure 14-16 Alternative splicing of SV40 T antigen Splicing out intron for Large T Splicing out intron for small t 28

Mutually exclusive splicing: Steric(입체적) hindrance(방햬) 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 Figure 14-17 U1-snRNP Mutually exclusive splicing: Steric hindrance 29

Mutually exclusive splicing: Figure 14-18 Mutually exclusive splicing: Combination of major and minor splice sites Non-sense mediated decay Figure 14-18 Combination of major and minor splice sites Non-sense mediated decay 30

Figure 14-19 Curious Drosophila Dscam (down syndrome cell adhesion molecule) gene: Mutually exclusive splicing on a grand scale (encode 38,016 protein sioforms) Figure 14-19 Curious Drosophila Dscam (down syndrome cell adhesion molecule) gene: Mutually exclusive splicing on a grand scale 31

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) 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) 32

Figure 14-21 Figure 14-21 33

Box 14-3-1 Box 14-3-1 34

Box 14-3-2 Box 14-3-2 35

Figure 14-22 Alternative (대체)splicing(이어맞추기) is regulated by activators and repressors. Figure 14-22 Alternative splicing is regulated by activators and repressors. 36

Figure 14-22a Exonic (or intronic) splicing silencers (ESS or ISS): sites that repressor protein binds Figure 14-22a 37

Figure 14-22b SR protein has RMM (RNA recognition motif domain) and RS domain (rich in arginine and serine) Exonic (or intronic) splicing enhancers (ESE or ISE): sites that SR protein binds (SR protein) Figure 14-22b Exonic (or intronic) splicing enhancers (ESE or ISE): sites that SR protein binds Exonic (or intronic) splicing silerncers (ESS or ISS): sites that repressor protein binds SR protein has RMM (RNA recognition motif domain) and RS domain (rich in arginine and serine) 38

Two mechanisms of silencer action Figure 14-23 Two mechanisms of silencer action A: A1(repressor) competes off SC35(activator) after binding to ESS. 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. silencer enhancer (repressor) Heterogeneous nuclear ribonucleoprotein (hnRNP) Figure 14-23 Two mechanisms of silencer action A: A1(repressor) compete of SC35(activator) after binding to ESS. B: PTB(hnRNP, Pyrimidine binding protein) interact with U1 at the 5’ splicing site and then block U1 to interact with 3’ splicing site. hetergenous nuclear rinonucleoprotein (RNP) 39

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 Figure 14-24 Regulation of alternative splicing determines the sex of flies Sxl: sex lethal Sis-a and sis-b: Sxl activators Dpn (Deadpan): sxl repressor X means X chromosome A means autosome Pe: promoter for establishment Pm: promoter for maintenance Pe: promoter for establishment Pm: promoter for maintenance Sxl: sex lethal 40

Figure 14-25 Figure 14-25 Dsx: double sex gene Dsx: double sex gene 41

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 (protein encode by 17-18a-19 mRNA ) Figure 14-26 An alternative splicing switch lies at the heart of pluripotency Induced pluripotency stem cells (iPS cells) FOXP1 (Transcription factor) FOXP1-ES activates genes OCT4 and NANOG, etc. Induced pluripotent stem cells (iPS cells) 42

Exon shuffling Shuffle: mix cards (poker game) 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. Shuffle: mix cards (poker game)

Exons encode protein domains. Figure 14-27 Exons encode protein domains. Borders between exons and introns within a gene coincide with boundaries between domains. Figure 14-2 Exons encode protein domains. Exon shuffling Exon are shuffled by recombination to produce genes encoding new proteins. Intron early model Intron late model 44

Genes made up of parts of other genes Figure 14-28 Genes made up of parts of other genes Figure 14-28 Genes made up of parts of other genes 45

Figure 14-29 Accumulation, loss, and reshuffling of domains during the evolution of a family of proteins Figure 14-29 ©2001 Macmillan www.nature.com Accumulation, loss, and reshuffling of domains during the evolution of a family of proteins 46

RNA editing is another way of altering the sequence of an mRNA Figure 14-30 RNA editing is another way of altering the sequence of an mRNA Stop codon Figure 14-30 RNA editing is another way of altering the sequence of an mRNA Stop codon 47

RNA editing by deaminase Figure 14-31 RNA editing by deaminase Figure 14-31 RNA editing by deaminase ADAR (adenosine deaminase acting on RNA) Inosine base pair with cytosine Inosine base pair with cytosine. So can alter sequence of protein ADAR (adenosine deaminase acting on RNA) 48

Figure 14-32 Figure 14-32 RNA editing by guide RNA-mediated U insertion (in trypanosome coxII gene RNA) RNA editing by guide RNA-mediated U insertion (in trypanosome coxII gene RNA) 49

Figure 14-32a Figure 14-32a 50

Figure 14-32b Figure 14-32b 51

Figure 14-32c Figure 14-32c 52

Figure 14-33 Transport of mRNAs (active transport) out of the nucleus through nuclear pore complex Figure 14-33 Transport of mRNAs out of the nucleus Less than 50kDa could pass without any help. 53