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Lecture 12

Lecture 12. Chapter 5 mRNA Modifica- tions in Eukaryotes 5.1 Capping 5.2 Polyadenylation 5.3 Splic ing 5.4 mRNA Editing 5.5 Experiments. 第 5 章 真核生物 mRNA 的修饰 5.1 加帽 5.2 聚腺苷酸化 5.3 剪 接 5.4 mRNA 编辑 5.5 实验研究.

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Lecture 12

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  1. Lecture 12 Chapter 5 mRNA Modifica- tions in Eukaryotes5.1 Capping 5.2 Polyadenylation 5.3 Splicing 5.4 mRNA Editing 5.5 Experiments 第5章 真核生物 mRNA的修饰 5.1 加帽 5.2 聚腺苷酸化 5.3剪接 5.4 mRNA编辑 5.5 实验研究

  2. Eukaryotic transcriptional products: used directly? 真核生物转录产物：直接使用吗？

  3. Questions for Lecture 12 Q1→ How is the cap added?帽子是怎么加上去的? Q2→ What functions does the cap have?帽子有什么作用? Q3→ How is a poly(A) tail added?Poly(A)尾是怎么加上去的? Q4→ What functions does the poly(A) tail have? Poly(A)尾有什么作用? Q5→ What is splicing? 什么是剪接？ Q6→ How is an intron removed?内含子是怎么去除的？

  4. Vocabulary of Lecture 12 (1/2) modification capping degrade, degradation polyadenylation poly(A) tail intron exon splicing splice site spliceosome 修饰（作用） 加帽（过程） 降解 聚腺苷化（作用） 聚腺苷酸尾 内含子 外显子 剪接（作用） 剪接位点 剪接体

  5. Vocabulary of Lecture 12 (2/2) lariat snRNPs RNA triphosphatase guanylyl transferase methyl transferase 套索 核内小核糖核蛋白 RNA三磷酸酶 鸟苷酸转移酶 甲基转移酶

  6. Q1→ How is the cap added?帽子是怎么加上去的? How can I get a nice hat for my daughter?

  7. 5.1 Capping / 加帽 加帽：将鸟嘌呤核苷的衍生物加到前体mRNA 5’端上去的转录后修饰作用。 Capping:Post-transcriptional modification in which a derivative of guanosine is attached to the 5’ end of the pre-mRNA. 前体mRNA：真核mRNA的前体，已经被转录出来但还没有经过转录后修饰 Pre-mRNA:The precursor to a eukaryotic mRNA, which has been transcribed but has not yet undergone post-transcriptional modifications.

  8. Structure of the cap / 帽的结构 5’-3’ phospho- diester bond 5’-5’ triphosphate bond

  9. RNA polymerase II Discussed in Chapter 4

  10. Capping takes place quite early 5’-Cap RNA DNA RNA polymerase II mRNA的5’端最先被转录出来，对5’端的加帽工作在基因的其它部分还没有被转录出来的时候就完成了。

  11. A1→ Capping process / 加帽过程 甲基转移酶 RNA三磷酸酶 鸟苷酸转移酶

  12. CTD: C-terminal domain RNA triphosphatase Methyl transferase Guanylyl transferase DNA RNA polymerase II 这三种酶在RNA聚合酶II开始转录前体mRNA的时候聚集在它的Rpb1亚基的C末端功能域中。这一功能域被称为‘该CTD’。

  13. Q2→ What functions does the cap have?帽子有什么作用? Can you suggest functions for the cap of an mRNA?

  14. A2→ Functions of the cap structure帽结构的功能 1. Helps prevent degradation帮助防止降解 2. Helps transport into cytoplasm帮助转运到细胞质中 3. Enhances translation / 增强转译 4. Helps remove the first intron帮助去除第一个内含子

  15. 1. Helps prevent degradation帮助防止降解 RNase RNase 5’-3’ phospho diester bond 5’-3’ phospho diester bond 5’-5’ triphosphate bond 核糖核酸酶一般不能降解帽结构中的三磷酸键

  16. 2. Helps transport into cytoplasm帮助转运到细胞质中 Discussed in Chapter 7

  17. 3. Enhances translation / 增强转译 Cap-binding protein No translation occurs. 为了与核糖体结合, mRNA需要帽结合蛋白的帮助。

  18. 4. Helps remove the first intron帮助去除第一个内含子 The first intron RNA DNA RNA polymerase II

  19. 5.2 Polyadenylation / 聚腺苷酸化 聚腺苷酸化：一种修饰过程，将一串长度约为250个腺嘌呤核苷酸加到前体mRNA的3’端。 Polyadenylation:A modification process in which a string of about 250 adenine nucleotides is added to the 3’ end of a pre-mRNA. Pre-mRNA AAAAAAA - - - - - - AAAA Poly(A) tail

  20. Q3→ How is a poly(A) tail added?Poly(A)尾是怎么加上去的? Squirrel 松鼠 “After your ‘tail bud’ is removed.” “Mum, how can I have a tail like you?”

  21. Polyadenylation does not occur at the natural end 聚腺苷酸化不是在自然尾部进行的 要将前体mRNA上先切去一小段之后poly(A)尾才加上去

  22. The polyadenylation signal / 聚腺苷酸化信号 Polyadenylation signalhas a typical sequence of AAUAAA. This sequence gives an instruction as “to cut the mRNA about 20 nucleotides downstream, near a GU-rich sequence”. Pre-mRNA AAUAAA GU Polyadenylation AAAAAAA - - - - - - AAAA Poly(A) tail

  23. The cleavage complex / 切割复合体 CPSF (cleavage and polyadenylation specificity factor) Cleavage complex CstF (cleavage stimulation factor) CFI (cleavage factor I) CFII (cleavage factors II)

  24. A3→ The polyadenylation mechanism 聚腺苷酸化机理 What are the possible reasons for polyade-nylation not to occur at the natural end ? 聚腺苷酸化不在自然尾部进行的可能原因是什么？ Poly(A)-Binding Protein

  25. CTD: C-terminal domain Proteins for capping at CTD Proteins for polyadenylation at CTD 加帽完成后，CTD被逐步磷酸化，原先存在于CTD周围的加帽蛋白逐渐离去，以便让聚腺苷酸化蛋白取代它们的位置。

  26. Q4→ What functions does the poly(A) tail have? Poly(A)尾有什么作用? Squirrel 松鼠 “Well, that is because … ” “Mum, why should I have the tail?”

  27. A4-1→ Functions of the poly(A) tailpoly(A)尾的功能 The main function of poly(A) tail is to protect the mRNA from degradation by ribonucleases. AAAAAAA - - - - - - AAAA A AAAAAAA - - - A A A A A

  28. A4-2→ Functions of the poly(A) tailpoly(A)尾的功能 There is also some evidence that the poly-A tail is involved insplicing and enhances translation of mRNAs. AAAAAAA - - - - - - AAAA

  29. Q5→ What is splicing? 什么是剪接？ E. coli: Ha ha ha, those eukaryotes have genes in pieces. It must bring a lot of trouble to paste them together…

  30. A5→ 5.3 Splicing / 剪接 Splicing: The process of removing introns from a pre-mRNA. 剪接：将内含子从前体mRNA去除的过程。

  31. Introns and Exons / 内含子与外显子 Introns: Sequences that do not code for protein and interrupt the coding regions. Exons: Parts of a gene that are expressed as protein. Exons are formed by the interruption of coding regions by introns. 内含子：不编码蛋白质并干涉打断编码区的序列。 外显子：基因中被表达成蛋白质的部分。由内含子打断编码区域而形成。

  32. Q6→ How is an intron removed?内含子是怎么去除的？ DNA Exon Intron Working copy of gene Enzymes cut out introns and splice exons together Molecule used to make protein

  33. 5.3.1 The Basic Splicing Reaction基本的剪接反应 剪接位点：表明内含子和外显子开始和结束位置的序列。 Splice sites:Sequences that mark the beginnings and ends of introns and exons. Splice sites in yeast 5’AG/GUAUGU…body of intron…UACUAAC-YAG/ 3’

  34. The basic splicing reaction 首先，内含子中一个核苷酸A上的羟基“进攻” 5’剪接位点。结果内含子变成了一个环，称为套索。 其次，外显子末端G上的羟基开始进攻3’剪接位点。 最后，在5’外显子和3’外显子之间形成新的键.

  35. 5.3.2 Proteins involved in Splicing在剪接中发挥作用的蛋白质 Spliceosome:The collection of factors, especially snRNPs, that help with the splicing of introns. : 剪接体：各种因子的集合体，尤其是snRNPs，帮助将内含子剪接掉。

  36. snRNPs snRNPs(核小核糖核蛋白)：是由RNA（核糖核酸）和蛋白质在细胞核中组成的大分子，在大多数内含子的剪接中都需要。 snRNPs (small nuclear ribonucleoproteins): macromolecules composed of RNA (ribonucleic acid) and protein that are found in the nucleus. They are required for splicing of most introns.

  37. snRNPs functioning in splicing在剪接中发挥作用的snRNPs snRNPs functioning in splicing include U1, U2, U4, U5 and U6. Why isn’t U3 here?

  38. A6→ The splicing reaction / 剪接反应

  39. Questions for extensive studies 用于拓展学习的问题 Q1 → Do rRNA and tRNA have 5’ cap, 3’ poly(A) tail and intron structures? Q2 → If they have, how are they added or removed? Q1 → rRNA和tRNA有没有5’帽、3’ poly (A)尾和内含子结构呢？ Q2 →如果有的话，它们是怎么加上去或去除的呢？

  40. Now showingmRNA splicing File: biophoto7 \Lehninger Principles of Biochemistry \ mRNA splicing

  41. mRNA splicing (1/8) Pre-mRNAs contain short, conserved sequences required for splicing. The most conserved intron sequences are the (5’)GU, (3’)AG, and the branch point A.

  42. mRNA splicing (2/8) Central to the splicing reactions are five small nuclear RNAs (snRNAs), complexed with proteins in small ribonucleoprotein particles (snRNPs). Additional proteins and ATP are also required for splicing, but are not shown here.

  43. mRNA splicing (3/8) The snRNAs bear base pair with pre-mRNA sequences and with each other to direct the splicing cycle. First, the 5’ end of U1 snRNA base pairs with the 5’ splice site, and a U2 snRNA sequence base pairs with the branch point region.

  44. mRNA splicing (4/8) Extensive base pairing between snRNAs in the U4 and U6 snRNPs forms a complex that associate with U5 snRNP. The U4/U6/U5 complex then associates with the pre-mRNA.

  45. mRNA splicing (5/8) Rearrangement of RNA-RNA base pairing occurs, so that U6 dissociates from U4 and base pairs with U2. U1 dissociates from the 5’ splice site, and U5 base pairs with exon sequences.

  46. mRNA splicing (6/8) The rearranged spliceosome catalyzes two transesterification reactions（转酯反应）, resulting in intron removal and exon ligation.

  47. mRNA splicing (7/8) The ligated exons are released from the spliceosome.

  48. mRNA splicing (8/8) The snRNPs dissociated from the excised lariat intron and are recycled for another round of splicing. The lariat intron is rapidly degraded.

  49. Now showing again

  50. Essentials of Lecture 12 / 第12讲要点 A1→ Capping process / 加帽过程 A2→ Functions of the cap structure帽结构的功能 A3→ The polyadenylation mechanism 聚腺苷酸化机理 A4→ Functions of the poly(A) tailpoly(A)尾的功能 A5→ Splicing / 剪接 A6→ The splicing reaction / 剪接反应

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