1.   Li Xiaoling Office: M1623 QQ: 313320773 E-MAIL: 313320773 @qq.com

2. Content Chapter 1 Introduction Chapter 2 The Structures of DNA and RNA Chapter 10 R egulation in Eukaryotes Chapter 4 DNA Mutation and Repair Chapter 5 RNA Transcription Chapter 6 RNA Splicing Chapter 7 Translation Chapter 8 The Genetic code  Chapter 9 R egulation in prokaryotes Chapter 3 DNA Replication

3. HOW TO LEARN THIS COURSE WELL ？  To learn effectively  To preview and review  Problem-base learning  Making use of class time effectively  Active participation  Bi-directional question in class  Group discussion  Concept map  Tutorship  To call for reading, thingking and discussing of investigative learning 2015-9-13

4. E VALUATION ( GRADING ) SYSTEM  Question in-class and attendance : 10 points  Group study and attendance: 20 points  Final exam: 70 points  Bonus 2015-9-13

5. Ch 5 : Transcription Ch 6 : RNA Splicing Ch 7 : Translation Ch 8 : The Genetic code 4/3/05

6. CHAPTER 6 RNA Splicing Molecular Biology Course

7. Figure 6-1 Primary transcript

8. 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.

9. Topic 1 : THE CHEMISTRY OF RNA SPLICING CHAPTER 6 RNA Splicing

10. S EQUENCES WITHIN THE RNA D ETERMINE W HERE S PLICING O CCURS The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs. The chemistry of RNA splicing

11. Figure 6-2 The consensus sequences for human

12. 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).

13. 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

14. Figure 6-3 Three-way junction

15. The structure of three-way junction Figure 6-4 This figure has an error Intron 5’ end

16. 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.

17. Figure 6-3

18. E XONS FROM DIFFERENT RNA MOLECULES CAN BE FUSED BY T RANS - SPLICING Trans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. The chemistry of RNA splicing

19. Trans-splicing Figure 6-5 Not a lariat

20. Topic 2 THE SPLICESOME MACHINERY THE SPLICESOME MACHINERY CHAPTER 6 RNA Splicing

21. 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

22. 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

23. 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.

24. Figure 6-6 RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA

25. Topic 3 SPLICING PATHWAYS CHAPTER 6 RNA Splicing

26. A SSEMBLY, REARRANGEMENT, AND CATALYSIS WITHIN THE SPLICEOSOME : THE SPLICING PATHWAY (F IG. 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

27. 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.

28. Figure 6-8 E complex A complex Figure 6-6b

29. 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.

30. Figure 6-8 A complex B complex

31. 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.

32. Figure 6-8 Figure 6-6c B complex C complex in which the catalysis has not occurred yet

33. 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.

34. 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

35. Figure 6-8 C complex 1 st reaction 2 nd reaction

36. E complex A complex B complex C complex （没有 该 complex 的图） splicesome-mediated splicing reactions Figure 6-8

37. H OW 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.

38. Figure 6-12

39. 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.

40. (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.

41. 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

42. 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

43. SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites. Figure 6-13

44. 1. Ensure the accuracy and efficacy of constitutive splicing ( 组成性剪接 ). 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 essential for splicing

45. T OPIC 4 ALTERNATIVE SPLICING CHAPTER 6 RNA Splicing

46. S INGLE GENES CAN PRODUCE MULTIPLE PRODUCTS BY ALTERNATIVE SPLICING 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. Alternative splicing

47. Drosophila DSCAM gene can be spliced in 38,000 alternative ways Figure 6-13

48. Figure 6-15 D IFFERENT WAYS OF ALTERNATIVE SPLICING Figure 6-14

49. 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 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

50. An example of constitutive alternative splicing : Splicing of the SV40 T antigen RNA Figure 6-16

51. A LTERNATIVE SPLICING IS REGULATED BY ACTIVATORS AND REPRESSORS The regulating sequences : exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). Alternative splicing Activators are proteins bind to enhancers to enhance splicing. Repressors are proteins bind to silencers to repress splicing.

52. SR proteins are splicing activators and contain two domains. (1) One domain is the RNA-recognition motif (RRM), which is responsible for RNA binding. (2) 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.

53. hnRNPs are splicing 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.

54. Regulated alternative splicing Figure 6-17

55. Binds at each end of the exon and conceals ( 隐藏 ) it Coats the RNA and makes the exons invisible to the splicing machinery Two models for the action of a repressor hnRNPI/PTB in inhibiting splicing Figure 6-18

56. The outcome of alternative splicing ( 可变剪接的结果 / 生 物学功能 ) 1. Producing multiple protein products, called isoforms. They can have similar, distinct or antagonistic functions. [One gene encodes multiple functions] 2. Switching 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]

57. A SMALL GROUP OF INTRON ARE SPLICED BY MINOR SPLICEOSOME It splices introns harboring determinant sequences distinct from those recognized by the major spliceosome. It is known AT-AC spliceosome. The termini of the originally identified introns that is splice contain AU at 5’ss, and AC at the 3’ ss. The chemical pathway is the same as the major spliceosome, but U11 and U12 are used in places of U1 and U2, respectively. Alternative splicing

58. Figure 6-19 The AT-AC spliceosome

59. Figure 6-20 Sequences conserved in different kinds of introns. Trans-splicing

60. Topic 5 : Self-splicing introns CHAPTER 6 RNA Splicing

61. S ELF - SPLICING INTRONS REVEAL THAT RNA CAN CATALYZE SPLICING Self-splicing introns: ---Introns that can fold into a specific conformation within the precursor RNA, and catalyze the chemistry of their own release and the exon ligation. ---They can remove themselves from pre-RNAs in the absence of any proteins or other RNAs in vitro. Splicing pathways

62. fold into a specific conformation catalyze the chemical reaction using metal ions as cofactors

63. There are two classes of self- splicing introns: group I self-splicing introns group II self-splicing introns.

64. TABLE 6-1 Three classes of RNA Splicing ClassAbundanceMechanismCatalytic Machinery Nuclear pre- mRNA Very common; most eukaryotic genes Two sequential transesterification reactions; branch site A spliceosome Group II introns Rare; some eukaryotic genes from organelles and prokaryotes Same as pre-mRNARNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genes Two sequential transesterification reactions; exogenous G Same as group II introns

65. Figure 6-9 The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.

66. The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification Figure 6-10

67. G ROUP I INTRONS RELEASE A LINEAR INTRON RATHER THAN A LARIAT During the 1 st transesterification reaction, group I introns use a free G, instead of using a branch point A, to attack the 5’ splice site. As a result, this G is attached to the 5’ end of the intron. A 3’-OH group is resulted at the 5’ exon, which then attacks the 5’ splice site for the 2 nd transesterification reaction. This is the same as that of splicing of the group II and pre-mRNA introns. Splicing pathways

68. G instead of A a linear intron a Lariat intron Figure 6-9

69. 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. Their tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor. Group I introns

70. Adams et al., Nature 2004, Crystal structure of a self- splicing group I intron with both exons. Group I intron website: www.rna.whu.edu.cn/gissdwww.rna.whu.edu.cn/gissd 2D structure 3D structure

71. T OPIC 6 RNA EDITING CHAPTER 6 RNA Splicing

72. RNA EDITING IS ANOTHER WAY OF CHANGING THE SEQUENCE OF AN M RNA AT THE RNA LEVEL I. Site specific deamination ( 位点特异性去氨反 应 ): 1. A specifically targeted C residue within mRNA is converted into U by the deaminase ( 脱氨酶 ). The process occurs only in certain tissues or cell types and in a regulated manner. RNA editing

73. Figure 6-25

74. Stop code In liverIn intestines Figure 6-25 RNA editing by deamination. The human apolipoprotein gene

75. 2. 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.

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

77. 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

78. Figure 6-26 RNA editing by gRNA-mediated U insertion

79. T OPIC 7 M RNA TRANSPORT CHAPTER 6 RNA Splicing

80. O NCE PROCESSED, M RNA IS PACKAGED AND EXPORTED FROM THE NUCLEUS INTO THE CYTOPLASM FOR TRANSLATION All the fully processed mRNAs are transported to the cytoplasm for translation into proteins mRNA transport

81. 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 including SR protein that identifies it as being ready for transport. 2. Export takes place through the nuclear pore complex.

82. 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.

83. Figure 6-27

84. Maniatis and Reed, Nature 2002, 416:499-506 补充材料补充材料

85. 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 of the chapter