1、Chapter 5 mRNA Modifications in Eukaryotes 第5章 真核生物mRNA的 修饰,In prokaryotes, transcription produces a nearly exact mRNA copy of the DNA, and the transcript is immediately translated into protein. In eukaryotes, a series of modifications occur to mRNA during and after transcription.,在原核生物中，转录产生的mRNA几乎
2、是DNA的准确拷贝，并且这一转录产物会立即被转译成蛋白质。在真核生物中，转录时以及转录后会对mRNA进行一系列修饰。,mRNA Modifications in Eukaryotes,5.1 Capping 5.2 Polyadenylation 5.3 Splicing 5.4 mRNA Editing 5.5 Experiments,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,Chapter 5 mRNA Modifications in Eukaryotes,concept,The remove of nucleotides by both e
3、ndonucleases and exonucleases. The addtion of nucleotides to the 5-or 3-ends of the primary transcription or their cleavage product.,A primary transcript or pre-mRNA is an mRNA that has been transcribed but is not yet ready for translation.,RNA processing is the collective term used to describe thes
4、e alterations to the primary transcript.,5.1 Capping / 加帽,Capping is the process of adding a derivative(m7G) of guanine nucleotide to the 5 end of the pre-mRNA.,This derivative is attached to the 5 end of the pre-mRNA by a 5-5 triphosphate bond linkage. The reaction is carried out by an enzyme calle
5、d guanyltransferase(鸟苷转移酶).,Structure of the cap / 帽的结构,5-3 phospho- diester bond,5-5 triphosphate bond,7-methylguanine nucleotide,methyl group,Capping takes place quite early.,Transcription of an mRNA occurs in the 5 to 3 direction. So, the 5 end of the mRNA comes out first. Capping takesplace quit
6、e early, before the rest of the gene is transcribed.,Capping process,Inverted guanine nucleotide,CTD: C-terminal domain,These three enzymes stay together around the C-terminal domain of the Rpb2 subunit of RNA polymerase II. They act together to complete the capping process.,Functions of the cap str
7、ucture,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,1. Helps prevent degradation,RNAs in the cell can be rapidly degraded by ribonucleases. However, these enzymes are generally not capable of d
8、egrading the triphosphate bond in the cap structure.,2. Helps transport into cytoplasm,Cap structure helps the RNA transcript to pass through selective pores of the nuclear membrane and into the cytoplasm.,3. Enhances translation / 增强转译,In order to bind the ribosome, mRNA requires help from cap-bind
9、ing protein. this protein requires the presence of the cap structure.,ribosome,4. Helps remove the first intron,Cap is required for mRNA splicing, to occur completly. Specifically, its presence is required for splicing of the first intron.,AAAAAAA - - - - - - AAAA,5.2 Polyadenylation / 聚腺苷酸化,Poly(A)
10、 tail,Polyadenylation,Pre-mRNA,Polyadenylation is a modification process in which a string of about 250 adenine nucleotides is added to the 3 end of the transcript.,Polyadenylation signal / 聚腺苷酸化信号,AAAAAAA - - - - - - AAAA,Poly(A) tail,Polyadenylation,Pre-mRNA,Polyadenylation signal has a typical ty
11、pical sequence of AAUAAA. This sequence gives an instruction as “to cut the mRNA about 20 nucleotides downstream, near a GU-rich sequence”.,AAUAAA,GU,AAUAAA motif by itself not sufficient for polyadenylation.,If it were, then polyadenylation would occur downstream of the many AAUAAA sequence found i
12、n introns, but it does not.,Polyadenylation Signals:,50-250 poly(A),Cleavage and polyadenylation of a pre-mRNA,polyadenylation involves both cleavage of the pre-RNA and polyadenylation at the cleavage site.,Requires several proteins: CPSF （切割与聚腺苷酸化特异因子）, CstF （切割激活因子）, CFI , CFII （切割因子I和II）, poly(A)
13、polymerase, and RNA polymerase II(in particular , the CTD of RPb2).,The cleavage complex / 切割复合体,Cleavage complex,CPSF (cleavage and polyadenylation specificity factor),CstF (cleavage stimulation factor),CFI (cleavage factor I),CFII (cleavage factors II),AAUAAA signal has been transcribed, CPSF bind
14、s to the AAUAAA sequence.,CstF binds to the G-U rich sequence,PBP can help poly(A) polymerase to work more efficiently.,CTD: C-terminal domain,Proteins for capping at CTD,Proteins for tailing at CTD,after capping is performed, CTD will be phosphorylated Gradually and the capping proteins are removed
15、.,Functions of the poly(A) tail poly(A)尾的功能,AAAAAAA - - - - - - AAAA,AAAAAAA - - -,A,A,A,A,A,A,The main function of poly(A) tail is to protect the mRNA from degradation by ribonucleases.,It depends on the long string of As that separate thecoding region from the end of the mRNA,AAAAAAA - - - - - - A
16、AAA,There is also some evidence that the poly-A tail is involved in splicing and enhances translation of mRNAs.,5.3 Splicing / 剪接,Exons: Parts of a gene that are expressed as protein.,Introns: Sequences that do not code for protein and interrupt the coding regions.,Splicing: The process of removing
17、introns and rejoin the exon from a pre-mRNA.,Interrupt gene,Junction sequence of intron linking with exon 高度保守，成为剪接过程重要的识别序列。 人类许多重要疾病的病因Junction seq. mut. 异常剪接 病症 地中海贫血症： junction sequence mutation of globin gene 干扰mRNA成熟,Junction sequence,5.3.1 The Basic Splicing Reaction 基本的剪接反应,5 AG/GUAUGUbody o
18、f intronUACUAAC-YAG / 3,Splice sites in yeast,Splice sites: Sequences that mark the beginning and ends of introns.,Almost all introns have GU on 5 end and have AG on 3 end. This typical structure forms the splice sites for splicing reaction.,5 splicing site or donor site,3 splicing site or acceptor
19、site,核内前体mRNA splicing,Group introns,Lariat inron,Group splicing model,Hydroxyl group on the A attacks the 5 splice site.,The bond between G of the exon and the G of intron at the boundary is broken.,The -OH of the exon terminal G attacks the 3 splice site.,Removal of the intron is complete.,Protein
20、s 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.,These proteins form spliceosome.,snRNPs核内小核糖核蛋白,剪接体,snRNPs: small nuclear ribonucleoproteins,snRNPs are small particles found in the nucleus and contain both protein
21、 and RNA （SnRNA）.,snRNPs functioning in splicing: U1, U2, U4, U5 and U6.,SnRNA can form specificbase pairs with the pre-mRNA.,U1 binds at the 5 splice site. Next, U2 binds at the attacking A.,Splicing process of spliceosome,U4 and U6 join the 5 splice site while U5 holds the 5 and 3 splice sites tog
22、ether.,Finally, U6 and U2 work together to carry out the major reactions of splicing.,U4 and U6 bind to each other.,Next, U4 unbinds U6. This activates U6, and it removes U1 from the 5 splice site.,5.3.2 Self-Splicing / 自我剪接,Tetrahymena thermophilia 嗜热四膜虫,Splicing that occurs without the help of pro
23、teins or snRNPs is called self-splicing.,低等真核生物rRNA，线粒体和叶绿体基因内含子。 Intron 含有多个保守的Central Core Seqence（中 部核心结构）它们反向平行，序列互补，形成分子 内二级结构。 Internal guide sequence (IGS): 与5donor，3acceptor序列互补，使U与G靠近。,Self-splicing of group I intron,IGS of tetrahymena rRNA,IGS：内含子内存在的一段序列，可以与5供点或3受点边界序列互补。,IGS保守序列：GGAGGG,C
24、CS,Reaction of Self-splicing,5 splice site is broken by attack from a G.,The free end of the exon attacks the 3 splice site, displacing the intron and forming a new bond with the next exon.,二次转酯反应,413nt intron,15nt,Ribozyme(核酶)：象35srRNA具有酶活性，能自我催化完成剪接的RNA。,IGS,Self-splicing of group II intron,核mRNA，
25、tRNA,攻击位点,牛仔套马索,5-2磷酸二酯键,two transesterification,5.3.4 Trans-Splicing / 反式剪接,被剪接的前导序列,cis-splicing,小外显子,-OH attack the 5 splice site,mRNA前体含有 Leading sequence,Y-内含子,5.3.5 Alternative splicing / 可变剪接,Alternative splicing: a kind of splicing that can produce various proteins(isoform protein) from one
26、gene.,constitutive,同源异型蛋白,By choosing various combinations of 17 exons from a8,000 different proteins can be produced from this one gene.,Drosophila Dscam gene,Exon 3,Exon 5,4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12,Exon 4 Exon 6 Exon 9,Alternative Splicing Goes Mainstream 可变剪接成为主流 The Scie
27、ntist, Volume 17(24): 28, Dec. 15, 2003,contains 116 exons,stick antibody in the membrane,Alternative splicing in immune cells,transmembrane region,secreted antibody,5.4 mRNA Editing / mRNA编辑,RNA编辑是指在mRNA水平上改变遗传信息的过程。指基因转录产生的mRNA分子中，由于核苷酸的缺失，插入或置换，基因转录物的序列不与基因编码序列互补，使翻译生成的蛋白质的氨基酸组成，不同于基因序列中的编码信息现象。,
28、mRNA编辑,通过编辑，可以给mRNA前体添加新的遗传信息。,Sleeping sickness is one of the most neglected human diseases, and mainly affects the most deprived peoples of some African countries.,Trypanosome 锥虫,Trypanosomes are protozoa that cause African sleeping sickness.,Editing mechanism,What determines where the editing sys
29、tem shoule add UMP?,Guide RNA is a small RNA whose partial sequence is complementary to the sequence of an RNA that will be edited.,Insertion editing-向导RNA介导的编辑：,指导RNA：一类小RNA分子，其部分序列 可与被编辑地RNA 序列互补。,At the 5-end, anchor sequence directs the gRNA to the region of the mRNA it will edit.,apolipoprotein
30、 B (APOB)（载脂蛋白）,The apolipoprotein B (APOB) carries cholesterol in the body. In the liver, a large version of APOB is needed, and the gene is transcribed and translated as normal.,In the small intenstine（小肠） a short version of APOB is needed.,CU editing is performed on one nucleotide of mRNA. The ch
31、ange of base causes a translation stop signal . The short version of the protein is produced.,5.5 Experiments / 实验研究,Hybridization between mRNA with DNA,The exons in the DNA hybridize to the mRNA. Because the mRNA does not contain introns, the introns in the DNA form large loops.,Detection of intron
32、s with DNase,Exposing the hybrid molecule to nucleases that only cut ssDNA. DNA in the loops was cut into little pieces.,The exon DNA, which hybridized to the mRNA, was protected.,by examining the DNA that was not cut, it was possible to determine which parts of the gene were exons.,Hybridization be
33、tween pre-mRNA and DNA,Were the introns even transcribed? Or were they transcribed and then removed?,Pre-mRNA hybridized perfectly to the DNA without leaving any loops.,This meant that introns were present in the pre-mRNA, and introns are indeed transcribed and then removed later.,1.Briefly describe the G capping reaction in eukaryotes. 简要描述真核生物的加帽反应。 2. Describe the process of tailing. 3.Compare splicing mechanisms of three types introns.,4.Which type of RNA is involved in the splicing of pre-mRNA molecules in eukaryotic cells?( ) A rRNA B hnRNA C snRNA D gRNA,Exercises,
