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1 Mechanisms of transcription RNA splicing Translation The genetic code Section III: Expression of the Genome.

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1 1 Mechanisms of transcription RNA splicing Translation The genetic code Section III: Expression of the Genome

2 2 Mechanisms of Transcription 1. RNA polymerase and transcription cycle 2. The transcription cycle in bacteria 3. Transcription in eukaryotes Molecular Biology Course

3 3 The Central Dogma DNA RNA PROTEIN Transcription Translation replication

4 4 Transcription is very similar to DNA replication but there are some important differences : 1.RNA is made of ribonucleotides 2.RNA polymerase catalyzes the reaction 3.The synthesized RNA does not remain base-paired to the template DNA strand 4.Less accurate (error rate: 10 -4 )

5 5 5.Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (Replication?)

6 6 Transcription of DNA into RNA Transcription bubble

7 7 Topic 1: RNA Polymerase and The Transcription Cycle Mechanisms of Transcription

8 8 RNA polymerases come in different forms, but share many features RNA polymerases performs essentially the same reaction in all cells Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III RNA polymerase and the transcription cycle

9 9 RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes RNA Pol I transcribe the large ribosomal RNA precursor gene RNA Pol II transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes

10 10 The subunits of RNA polymerases

11 11 The bacterial RNA polymerase The core enzyme alone synthesizes RNA    ’’ 

12 12   ’’  RPB3 RPB11 RPB2 RPB1 RPB6 RNAP Comparison The same color indicate the homologous of the two enzymes prokaryotic eukaryotic 

13 13 “Crab claw” shape of RNAP (The shape of DNA pol is__) Active center cleft

14 14 There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzyme’s active center cleft

15 15 Transcription by RNA polymerase proceeds in a series of steps Initiation Elongation Termination RNA polymerase and the transcription cycle

16 16 Initiation Promoter: the DNA sequence that initially binds the RNA polymerase The structure of promoter- polymerase complex undergoes structural changes to proceed transcription DNA at the transcription site unwinds and a “ bubble ” forms Direction of RNA synthesis occurs in a 5 ’ -3 ’ direction (3 ’ -end growing)

17 17 initiation Binding (closed complex) Promoter “ melting ” (open complex) Initial transcription

18 18 Elongation Once the RNA polymerase has synthesized a short stretch of RNA (~ 10 nt), transcription shifts into the elongation phase. This transition requires further conformational change in polymerase that leads it to grip the template more firmly. Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain

19 19 Termination After the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript. In some cells, termination occurs at the specific and well- defined DNA sequences called terminators. Some cells lack such termination sequences.

20 20 Elongation and termination Termination Elongation

21 21 Transcription initiation involves 3 defined steps 1. Forming closed complex 2. Forming open complex 3. Promoter escape RNA polymerase and the transcription cycle

22 22 The initial binding of polymerase to a promoter DNA remains double stranded The enzyme is bound to one face of the helix Closed complex

23 23 Open complex the DNA strand separate over a distance of ~14 bp (-11 to +3 ) around the start site (+1 site) Replication bubble forms

24 24 Stable ternary complex The enzyme escapes from the promoter The transition to the elongation phase Stable ternary complex =DNA +RNA + enzyme RNA polymerase and the transcription cycle

25 25 Topic 2 The transcription cycle in bacteria Mechanisms of Transcription

26 26 Bacterial promoters vary in strength and sequences, but have certain defining features The transcription cycle in bacteria

27 27,Holoenzyme=  factor + core enzyme In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?

28 28 The predominant  factor in E. coli is  70. Promoter recognized by  70 contains two conserved sequences (-35 and – 10 regions/elements) separated by a non-specific stretch of 17-19 nt. Position +1 is the transcription start site. Promoters recognized by E. coli  factor

29 29 bacterial promoter The distance is conserved  70 promoters contain recognizable – 35 and – 10 regions, but the sequences are not identical. 2. Comparison of many different promoters derives the consensus sequences reflecting preferred – 10 and – 35 regions

30 30 Consensus sequence of the -35 and -10 region

31 31 3.Promoters with sequences closer to the consensus are generally “ stronger ” than those match less well. (What does “ stronger ” mean?) 4.The strength of the promoter describes how many transcripts it initiates in a given time.

32 32 bacterial promoter Confers additional specificity UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase

33 33 bacterial promoter Another class of  70 promoter lacks a – 35 region and has an “ extended – 10 element ” compensating for the absence of – 35 region

34 34 The  factor mediates binding of polymerase to the promoter The transcription cycle in bacteria The  70 factor comprises four regions called  region 1 to  region 4.

35 35 regions of  Region 4 recognizes -35 element Region 2 recognizes -10 element Region 3 recognizes the extended – 10 element

36 36 Binding of –35 Two helices within region 4 form a common DNA-binding motif, called a helix-turn-helix motif Helix-turn-helix DNA-binding motif One helix inserts into the DNA major groove interacting with the bases at the – 35 region. The other helix lies across the top of the groove, contacting the DNA backbone

37 37 Interaction with –10 is more elaborate ( 精细 ) and less understood The -10 region is within DNA melting region The  helix recognizing – 10 can interacts with bases on the non-template strand to stabilize the melted DNA.

38 38 UP-element is recognized by a carboxyl terminal domain of the - subunit (CTD), but not by  factor  and  subunits recruit RNA pol core enzyme to the promoter

39 39 Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA This transition is called Isomerization ( 异构化 ) The transcription cycle in bacteria

40 40 For  70 –containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement)

41 41 the opening of the DNA double helix, called “ melting ”, at positions -11 and +3. Change of the promoter DNA

42 42 The striking structural change in the polymerase 1. the and ’ pincers down tightly on the downstream DNA 2. A major shift occurs in the N- terminal region of  (region 1.1) shifts. In the closed complex,  region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft

43 43 NTP uptake channel is in the back channels into and out of the open complex DNA entering channel

44 44 Transcription is initiated by RNA polymerase without the need for a primer Initiation requires: The initiating NTP (usually an A) is placed in the active site The initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme The transcription cycle in bacteria

45 45 RNA polymerase synthesizes several short RNAs before entering the elongation phase Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt. The transcription cycle in bacteria

46 46 Structural barrier for the abortive initiation The 3.2 region of  factor lies in the middle of the RNA exit channel in the open complex. Ejection of this region from the channel (1) is necessary for further RNA elongation, (2) takes the enzyme several attempts

47 47 NTP uptake channel is in the back channels into and out of the open complex DNA entering channel

48 48 The elongating polymerase is a processive machine that synthesizes and proofreads RNA The transcription cycle in bacteria

49 49 1. DNA enters the polymerase between the pincers 2. Strand separation in the catalytic cleft 3. NTP addition 4. RNA product spooling out (Only 8- 9 nts of the growing RNA remain base-paired with the DNA template at any given time) 5. DNA strand annealing in behind Synthesizing by RNA polymerase

50 50 Pyrohosphorolytic (焦磷酸键解) editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. Hydrolytic (水解) editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimulation factor. Proofreading by RNA polymerase

51 51 Transcription is terminated by signals within the RNA sequence Terminators: the sequences that trigger the elongation polymerase to dissociate from the DNA Rho-dependent (requires Rho protein) Rho-independent, also called intrinsic ( 内在 ) terminator The transcription cycle in bacteria

52 52 Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.

53 53 Weakest base pairing: A:U make the dissociation easier transcription termination

54 54 Rho () -dependent terminators Have less well-characterized RNA elements, and requires Rho protein for termination Rho is a ring-shaped single-stranded RNA binding protein, like SSB Rho binding can wrest ( 夺取 ) the RNA from the polymerase-template complex using the energy from ATP hydrolysis Rho binds to rut ( utilization) RNA sites Rho does not bind the translating RNA

55 55 the  transcription terminator Hexamer, Open ring RNA tread trough the “ring”

56 56 Topic 3: transcription in eukaryotes Mechanisms of Transcription

57 57 Comparison of eukaryotic and prokaryotic RNA polymerases Eukaryotes: Three polymerase transcribes different class of genes: Pol I-large rRNA genes; Pol II- mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6) Prokaryotes: one polymerase transcribes all genes

58 58 Comparison of eukaryotic and prokaryotic promoter recognition Eukaryotes: general transcription factors (GTFs). TFI factors for RNAP I, TFII factors for RNAP II and TFIII factors for RNAP III Prokaryotes: factors

59 59 In addition to the RNAP and GTFs, in vivo transcription also requires Mediator complex DNA-binding regulatory proteins chromatin-modifying enzymes Why??

60 60 RNA polymerase II core promoters are made up of combinations of 4 different sequence elements Eukaryotic core promoter (~40 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro The transcription in eukaryotes

61 61 TFIIB recognition element (BRE) The TATA element/box Initiator (Inr) The downstream promoter element (DPE) Pol II core promoter

62 62 The sequence elements other than the core promoter that are required to regulate the transcription efficiency Those increasing transcription: Promoter proximal elements Upstream activator sequences (UASs) Enhancers Those repressing elements: silencers, boundary elements, insulators ( 绝缘 体 ) Regulatory sequences

63 63 RNA Pol II forms a pre- initiation complex with GTFs at the promoter The involved GTFIIs (general transcription factor for Pol II) TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F, E, H The transcription in eukaryotes

64 64 1. TBP in TFIID binds to the TATA box 2. TFIIA and TFIIB are recruited with TFIIB binding to the BRE 3. RNA Pol II-TFIIF complex is then recruited 4. TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex 5. Promoter melting using energy from ATP hydrolysis by TFIIH ) 6. Promoter escapes after the phosphorylation of the CTD tail

65 65 Promoter escape Stimulated by phosphorylation of the CTD (C-terminal domain) tail of the RNAP II CTD contains the heptapeptide repeat Tyr-Ser-Pro-Thr-Ser-Pro-Ser Phosphorylation of the CTD “ tail ” is conducted by a number of specific kinases including a subunit of TFIIH

66 66 TBP binds to and distorts DNA using a sheet inserted into the minor groove Unusual (P367 for the detailed mechanism) The need for that protein to distort the local DNA structure The transcription in eukaryotes

67 67 A:T base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove

68 68 The other GTFs also have specific roles in initiation ~ 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA The transcription in eukaryotes

69 69 TFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA exit channel resembles the   TFIIB-TBP-promoter complex

70 70 TFIIF: (1) a two subunit factor, (2) binding of Pol II-TFIIF stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor binding TFIIE: recruits and regulates TFIIH TFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair.

71 71 in vivo, transcription initiation requires additional proteins The mediator complex Transcriptional regulatory proteins Nucleosome-modifying enzymes To counter the real situation that the DNA template in vivo is packed into nucleosome and chromatin The transcription in eukaryotes

72 72 assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators

73 73 Mediator consists of many subunits, some conserved from yeast to human More than 20 subunits 7 subunits show significant sequence homology between yeast and human Only subunit Srb4 is essential for transcription of essentially all Pol II genes in vivo Organized in modules ( 模块 ) The transcription in eukaryotes

74 74 comparison of the yeast and human mediators

75 75 Eukaryotic RNA Pol II holoenzyme is a putative preformed complex: Pol II + mediator + some of GTFs Prokaryotic RNA Polymerase holoenzyme: core polymerase +  factor

76 76 A new set of factors stimulate Pol II elongation and RNA proofreading The transcription in eukaryotes

77 77 Transition from the initiation to elongation involves the Pol II enzyme shedding ( 摆脱 ) most of its initiation factors (GTF and mediators) and recruiting other factors: (1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5. (2) RNA processing (RNA 加工 ) factors Recruited to the C-terminal tail of the CTD of RNAP II to phosphorylate the tail for elongation stimulation, proofreading, and RNA processing like splicing and polyadenylation.

78 78 RNA processing enzymes are recruited by the tail of polymerase

79 79 Some elongation factors P-TEFb: phosphorylates CTD Activates hSPT5 Activates TAT-SF1 TFIIS: Stimulates the overall rate of elongation by resolving the polymerase pausing Proofreading

80 80 Elongation polymerase is associated with a new set of protein factors required for various types of RNA processing RNA processing: Capping of the 5 ’ end of the RNA Splicing of the introns (most complicated) Poly adenylation ( 多聚腺苷化 ) of the 3 ’ end The transcription in eukaryotes

81 81 Evidence: this is an overlap in proteins involving in those events The elongation factor hSPT5 also recruits and stimulates the 5 ’ capping enzyme The elongation factor TAT-SF1 recruits components for splicing Elongation, termination of transcription, and RNA processing are interconnected/ coupled ( 偶联的 ) to ensure the coordination ( 协同性 ) of these events

82 82 Function of poly(A) tail Increased mRNA stability Increased translational efficiency Splicing of last intron

83 83 Function of 5´cap Protection from degradation Increased translational efficiency Transport to cytoplasm Splicing of first intron

84 84 RNA processing 1 5’ end capping The “ cap ” : a methylated guanine joined to the RNA transcript by a 5 ’ -5 ’ linkage The linkage contains 3 phosphates 3 sequential enzymatic reactions Occurs early

85 85 Splicing: joining the protein coding sequences Dephosphorylation of Ser5 within the CTD tail leads to dissociation of capping machinery Further phosphorylation of Ser2 recruits the splicing machinery

86 86 3’ end polyadenylation Linked with the termination of transcription The CTD tail is involved in recruiting the polyadenylation enzymes The transcribed poly-A signal triggers the reactions 1. Cleavage of the message 2. Addition of poly-A 3. Termination of transcription

87 87 1. CPSF (cleavage and polyadenylation specificity factor) & CstF (cleavage stimulation factor) bind to the poly-A signal, leading to the RNA cleavage 2. Poly-A polymerase (PAP) adds ~ 200 As at the 3 ’ end of the RNA, using ATP as a substrate polyadenylation and termination

88 88 What terminates transcription by polymerase?

89 89 Models to explain the link between polyadenylation and termination (see the animation on your CD) Model 1: The transfer of the 3 ’ processing enzyme to RNAP II induces conformational change — RNAP II processivity reduces — spontaneous termination Model 2: absence of a 5 ’ cap on the second RNA molecule — recognized by the RNAP II as improper — terminate transcription

90 90 RNA Pol I & III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP Pol I: transcribes rRNA precursor encoding gene (multi-copy gene) Pol III: transcribes tRNA genes, snRNA genes and 5S rRNA genes The transcription in eukaryotes

91 91 Pol I promoter recognition Pol I promoter region Upstream control element UBF binds to the upstream of UCE, bring SL1 to the downstream part of UCE. SL1 in turn recruits RNAP I to the core promoter for transcription

92 92 Pol III core promoter TFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP III Pol III promoter recognition 1. Different forms, 2. locates downstream of the transcription site

93 93 1.RNA polymerases (RNAP, 真核和原核的异同 ) and transcription cycle (Initiation is more complicate, details in bacteria) 2.Transcription cycle in bacteria: (1) promoters (elements),  factor (4 domains),  CTD, abortive initiation (why?) (2) Structures accounting for formation of the closed complex, transitions to open complex and then stable ternary complex. (3) Elongation and editing by polymerase (10 -4 ) (4) Termination: Rho-independent and Rho- dependent mechanism Key points of the chapter

94 94 3.Transcription cycle in eukaryotes: (1)Promoters (elements), general transcription factors (GTF), (2)RNAP II transcription ---the roles of GTFs and the CTD tail of RNAP II in promoter recognition, formation of the pre-initiation complex, promoter melting, promoter escape ---in vivo requires mediator complex, nucleosome modifying enzymes and transcription regulatory proteins. ---elongation and proofreading involve a new set of GTFs (What) ---coupled with RNA processing (How)

95 95 (3)RNAP I and III transcription ---GTFs and promoter recognition, formation of the initiation complex

96 96 RNA Splicing Molecular Biology Course

97 97 Primary transcript

98 98 Most of the eukaryotic genes are mosaic ( 嵌 合体 ), consisting of intervening sequences separating the coding sequence Exons ( 外显子 ): the coding sequences Introns ( 内含子 ) : the intervening sequences RNA splicing: the process by which introns are removed from the pre-mRNA. Alternative splicing ( 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.


100 100 Sequences within the RNA Determine Where Splicing Occurs The chemistry of RNA splicing The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.

101 101 The consensus sequences for human

102 102 5 ’ splice site (5 ’ 剪接位点 ): the exon- intron boundary at the 5 ’ end of the intron 3 ’ splice site (3 ’ 剪接位点 ): the exon-intron boundary at the 3 ’ end of the intron Branch point site ( 分枝位点 ): an A close to the 3 ’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

103 103 The intron is removed in a Form Called a Lariat ( 套马索 ) as the Flanking Exons are joined Two successive transesterification: 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 splicing

104 104 Three-way junction

105 105 The structure of three-way function This figure has an error

106 106 Step 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.

107 107

108 108 Exons from different RNA molecules can be fused by Trans-splicing Trans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. The chemistry of RNA splicing

109 109 Trans-splicing Not a lariat


111 111 RNA splicing is carried out by a large complex called spliceosome The above described splicing of introns from pre-mRNA are mediated by the spliceosome. The spliceosome comprises about 150 proteins and 5 snRNAs. Many functions of the spliceosome are carried out by its RNA components. The spliceosome machinery

112 112 The 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 “ snurps ” ). The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction

113 113 Three roles of snRNPs in splicing 1. Recognizing the 5 ’ splice 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.

114 114 RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA

115 115 Topic 3 SPLICING PATHWAYS RNA Splicing

116 116 Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway (Fig. 13-8) Assembly step 1 1. 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 branch point. 3. Early (E) complex is formed Splicing pathways

117 117 Assembly step 2 1. 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 ’ splice site.

118 118 E complex A complex

119 119 Assembly step 3 1. 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.

120 120 A complex B complex

121 121 Assembly step 4 U1 leaves the complex, and U6 replaces it at the 5 ’ splice site. U4 is released from the complex, allowing U6 to interact with U2 (Figure 13-6c).This arrangement called the C complex.

122 122 B complex C complex in which the catalysis has not occurred yet

123 123 Catalysis Step 1: C complex Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together 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 ( 转酯 ) reaction.

124 124 Catalysis Step 2: U5 snRNP 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 snRNPs

125 125 C complex 1 st reaction 2 nd reaction

126 126 E complex A complex B complex C complex (没有 该 complex 的图) splicesome-mediated splicing reactions

127 127 How does spliceosome find the splice sites reliably Splicing pathways Two kinds of splice-site recognition errors Splice sites can be skipped. “ Pseudo ” splice sites could be mistakenly recognized, particularly the 3 ’ splice site.

128 128

129 129 Reasons for the recognition errors (1) The average exon is 150 nt, and 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.

130 130 (2) The splice site consensus sequence are rather loose. For example, only AGG tri- nucleotides is required for the 3 ’ splice site, and this consensus sequence occurs every 64 nt theoretically.

131 131 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 are readily recognized, thus preventing exon skipping. Two ways to enhance the accuracy of the splice-site selection

132 132 2. 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 and promote the use of the nearby splice sites by recruiting the splicing machinery to those sites

133 133 SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.

134 134 1. Ensure the accuracy and efficacy of constitutive splicing 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 essential for splicing

135 135 Topic 4 ALTERNATIVE SPLICING RNA Splicing

136 136 Many 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. Single genes can produce multiple products by alternative splicing Alternative splicing

137 137 Drosophila DSCAM gene can be spliced in 38,000 alternative ways

138 138 There are five different ways to alternatively splice a pre-mRNA

139 139 Alternative splicing can be either constitutive or regulated Constitutive alternative splicing: more than one product is always made from a pre-mRNA Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types

140 140 An example of constitutive alternative splicing : Splicing of the SV40 T antigen RNA

141 141 Alternative splicing is regulated by activators and repressors The regulating sequences : exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). The former enhance and the latter repress splicing. Proteins that regulate splicing bind to these specific sites for their action Alternative splicing

142 142 SR proteins binding to enhancers act as activators. (1) One domain is the RNA-recognition motif (RRM) (2) The other domain is RS domain rich in arginine and serine. This domain mediates interactions between the SR proteins and proteins within the splicing machinery.

143 143 hnRNPs binds RNA and act as repressors 1. Most silencers are recognized by hnRNP ( 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.

144 144 Regulated alternative splicing

145 145 Binds at each end of the exon and conceals ( 隐藏 ) it Coats the RNA and makes the exons invisible to the splicing machinery An example of repressors: inhibition of splicing by hnRNPI

146 146 The outcome of alternative splicing: 1. Producing multiple protein products, called isoforms. 2. Switching on and off the expression of a given gene. In this case, one functional protein is produced by a splicing pattern, and the non- functional proteins are resulted from other splicing patterns.

147 147 A small group of intron are spliced by minor spliceosome This spliceosome works on a minority of exons, and those have distinct splice-site sequence. The chemical pathway is the same as the major spliceosome. Alternative splicing

148 148 The AT-AC spliceosome U11 and U12 are in places of U1 and U2, respectively

149 149 Topic x : Self-splicing introns 自剪接内含子 RNA Splicing

150 150 Self-splicing introns reveal that RNA can catalyze RNA splicing Self-splicing introns: the intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release and the exon ligation Splicing pathways

151 151 Adams et al., Nature 2004, Crystal structure of a self-splicing group I intron with both exons

152 152 Practical definition for self-splicing introns: the introns that can remove themselves from pre-RNAs in the test tube in the absence of any proteins or other RNAs. There are two classes of self- splicing introns, group I and group II self-splicing introns.

153 153 Three class of RNA Splicing ClassAbundanceMechanismCatalytic Machinery Nuclear pre- mRNA Very common; used for most eukaryotic genes Two transesterification reactions; branch site A Major spliceosome Group II introns Rare; some eu- Karyotic genes from organelles and prokaryotes Same as pre- mRNA RNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific- ation reactions; exogenous G Same as group II introns

154 154 The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.

155 155

156 156 Group I introns release a linear intron rather than a lariat Instead of using a branch point A, group I introns use a free G to attack the 5 ’ splice site. This G is attached to the 5 ’ end of the intron.The 3 ’ -OH group of the 5 ’ exon attacks the 5 ’ splice site. The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns. Splicing pathways

157 157 G instead of A a linear intron a Lariat intron

158 158 1. Smaller than group II introns 2. Share a conserved secondary structure, which includes an “ internal guide sequence ” base-pairing with the 5 ’ splice site sequence in the upstream exon. 3. The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor Group I introns

159 159 The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification

160 160

161 161 Topic 6 RNA EDITING RNA Splicing

162 162 RNA editing is another way of changing the sequence of an mRNA I. Site specific deamination : 1. A specifically targeted C residue within mRNA is converted into U by the deaminase. 2. The process occurs only in certain tissues or cell types and in a regulated manner. RNA editing

163 163

164 164 The human apolipoprotein gene Stop code In liverIn intestines

165 165 3. 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. 4. An ion channel expressed in mammalian brains is the target of Adenosine deamination.

166 166 II Guide RNA-directed uridine 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).

167 167 3. The addition of Us to the message changes codons and reading frames, completely altering the “ meaning ” of the message. 4. Us are inserted into the message by guide RNAs (gRNAs).

168 168 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 gRNAs

169 169

170 170 Topic 7 mRNA TRANSPORT RNA Splicing

171 171 Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation mRNA transport All the fully processed mRNAs are transported to the cytoplasm for translation into proteins

172 172 Movement from the nucleus to the cytoplasm is an active and carefully regulated process. The damaged, misprocessed and liberated introns are retained in the nucleus and degraded. 1. A typical mature mRNA carries a collection of proteins that identifies it as being ready for transport. 2. Export takes place through the nuclear pore complex.

173 173 3. Once in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.

174 174

175 175 1.Why RNA splicing is important? 2.Chemical reaction: determination of the splice sites, the products, trans-splicing 3.Spliceosome: splicing pathway and finding the splice sites 4.Self-splicing introns and mechanisms 5.Alternative splicing and regulation, alternative spliceosome 6.Two different mechanisms of RNA editing 7.mRNA transport-a link to translation Key points

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