RNA加工Post-transcriptional Processing ofmRNA Occurs in Nucleus

1、RNA的加工转录后的加工Post-transcriptional Processing ofmRNA Occurs in Nucleus1. Cap 帽子帽子2. poly A tail 尾巴尾巴3.Introns spliced out 剪除内含子剪除内含子0号帽子号帽子核糖不被甲核糖不被甲基化基化1号帽子号帽子一个核糖被一个核糖被甲基化甲基化2号帽子号帽子二个核糖二个核糖被甲基化被甲基化倒置的倒置的GTPPost-transcriptional Processing ofmRNA - Capping磷酸水解酶 鸟苷酸转移酶Post-transcriptional Processing of

2、mRNA - Methylating the Cap鸟嘌呤7-甲基转移酶 s-腺苷-1-高半胱氨酸 s-腺苷甲硫氨酸 Methylation of the 2-OH of First(and Second) Base Can Also OccurPost-transcriptional Processing ofmRNA - Polyadenylation核酸内切酶 聚腺苷酸聚合酶 Figure 13-1Primary transcript 多数真核基因都是断裂的，其编码序列由非编多数真核基因都是断裂的，其编码序列由非编码序列隔开码序列隔开 Exons (外显子外显子): the coding

3、 sequences Introns (内含子内含子) : 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.RNA的

4、序列决定剪接位点的序列决定剪接位点The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.剪接应该在那里发生？Figure 13-2The consensus sequences for humann5splice site (5剪接位点): the exon-intron boundary at the 5 end of the intronn3 splice site (3剪接位点): the exon-intron boundary at

5、the 3 end of the intronnBranch 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).两相邻的外显子连接起来，内含子以套两相邻的外显子连接起来，内含子以套马索马索(Lariat)的结构被除去的结构被除去两次两次转酯化反应(transesterification):Step 1: The OH of the conserved A at the branch site attacks the ph

6、osphoryl 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 splicingFigure 13-3Three-way junction 丁字路口结构 Three-way junctionFigure 13-4Step 2: The OH of the 5 exon attacks the phospho

7、ryl 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.Figure 13-3来至不同来至不同RNA分子的外显子可以通过反分子的外显子可以通过反式剪接式剪接(Trans-splicing)连接起来连接起来 Trans-splicing: the process in which two exons carried on different RNA molecules can be spli

8、ced together. Trans-splicingFigure 13-5Not a lariat剪接机器剪接机器RNA剪接由一个大的叫剪接体剪接由一个大的叫剪接体(spliceosome)的复合体执行的复合体执行 剪接体由大约剪接体由大约 150个蛋白和个蛋白和 5 snRNAs 组成组成 剪接体的许多功能都是由其剪接体的许多功能都是由其RNA组分执组分执行的行的 5 RNAs (U1, U2, U4, U5, and U6, 100-300 nt) 叫核内小叫核内小RNA(small nuclear RNAs, snRNAs). snRNA和蛋白的复合体叫和蛋白的复合体叫 核内小分子核

9、糖核核内小分子核糖核蛋白蛋白 small nuclear ribonucleoproteins (snRNP, 发音发音 “snurps”). 剪接体是最大的剪接体是最大的snRNP，在剪接的不同阶段，在剪接的不同阶段它的组成会有变化它的组成会有变化1. 识别识别 the 5 splice site and the branch site.2. 将这两个位点将这两个位点带带到一起到一起.3. 催化催化 (or 帮助催化帮助催化) RNA 断裂断裂.RNA-RNA, RNA-protein and protein-protein interactions are all important du

10、ring splicing.snRNPs 在剪接中的角色Figure 13-6RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNATopic 3：剪接途径剪接体的装配剪接体的装配, 重排重排, 和催化和催化 : the splicing pathway (Fig. 13-8)装配装配 step 11. U1 recognize 5 splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3 spli

11、ce site. The former subunits interacts with BBP and helps it bind to the branch point.3. Early (E) complex is formed装配装配 step 21. 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).

12、This A residue is available to react with the 5 splice site.Figure 13-8E complexA complexFigure 13-6b 装配装配 step 31. 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.Figure 13-8A complexB complex 装配装配 step 4U1 leaves the comp

13、lex, 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.Figure 13-8Figure 13-6cB complexC complex in which the catalysis has not occurred yet催化催化 Step 1:Formation of the C complex produces the

14、active site, with U2 and U6 RNAs being brought togetherFormation of the active site juxtaposes 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 reaction.催化 Step 2:U5 snRNP helps to bring the

15、two exons together, and aids the second transesterification reaction, in which the 3-OH of the 5 exon attacks the 3 splice site.n最后 Step: Release of the mRNA product and the snRNPsFigure 13-8C complexE complexA complexB complexsplicesome-mediated splicing reactionsFigure 13-8自我剪接内含子自我剪接内含子(Self-spli

16、cing introns)揭示揭示RNA可以催化可以催化RNA剪接剪接 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 Adams et al., Nature 2004, Crystal structure of a self-splicing group I intron with both exons

17、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. 有两类型的自我剪接内含子： group I and group II self-splicing introns.TABLE 13-1 RNA剪接的三种类型剪接的三种类型ClassAbundanceMechanismCatalytic MachineryNuclear p

18、re-mRNA很普遍很普遍; 被很多真核基因使被很多真核基因使用用两次转酯反应两次转酯反应; 分分支位点支位点 A大剪接体大剪接体Group II introns很少很少; 一些真核细胞器基因一些真核细胞器基因和原核基因和原核基因和和 pre-mRNA一一样样内含子编码的内含子编码的RNA酶酶 (核酶核酶)Group I introns很少很少; 一些真核细胞核一些真核细胞核 rRNA, 细胞器基因细胞器基因, 以及很以及很少的原核基因少的原核基因两次转酯反应两次转酯反应; 需需要外源的要外源的G参与参与和和 group II introns一样一样 II类内含子类内含子(group II i

19、ntron)剪接的化学剪接的化学反应及中间产物和核前体反应及中间产物和核前体RNA (nuclear pre-mRNA)一样一样.Figure 13-9I类内含子类内含子(Group I introns) 以线性形式释放以线性形式释放而非而非套马索形式套马索形式 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

20、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.G instead of Aa linear introna Lariat intronFigure 13-91. 比比 group II introns小小2. 有一个保守的二级结构，包含内在前导序有一个保守的二级结构，包含内在前导序列列 “internal guide sequence” ，该序列，该

21、序列可以和上游外显子的可以和上游外显子的5 剪接位点碱基配对剪接位点碱基配对.3. 三级结构有一个可以结合鸟苷酸或鸟三级结构有一个可以结合鸟苷酸或鸟甙的结合袋Group I intronsII 类内含子和 U2-U6 snRNA的结构相似性(在第一次转酯反应中)Figure 13-10剪接体怎样找到正确的碱基位点？剪接体怎样找到正确的碱基位点？两类碱基位点识别错误nSplice sites can be skipped.n“Pseudo” splice sites could be mistakenly recognized, particularly the 3 splice site. F

22、igure 13-12识别错误产生的原因(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. (2) The splice site consensus sequence are rather loose

23、. For example, only AGG tri-nucleotides is required for the 3 splice site, and this consensus sequence occurs every 64 nt theoretically. 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 RN

24、A ensures all the splice sites emerging from RNAP II are readily recognized, thus preventing exon skipping. 两条增加选择正确碱基位点的途径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 exo

25、ns and promote the use of the nearby splice sites by recruiting the splicing machinery to those sitesSR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites. Figure 13-131. Ensure the accuracy and efficacy of

26、constitutive splicing2. Regulate alternative splicing3. 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 Many genes in higher eukaryotes encode RNAs that can be

27、spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products.单基因可以通过选择性剪接产生多蛋白单基因可以通过选择性剪接产生多蛋白Drosophila DSCAM gene can be spliced in 38,000 alternative ways Figure 13-13Figure 13-15前体前体RNA的选择性剪接有的选择性剪接有5种不同的类型种不同的类型选择性剪接可以是组成性的，也可以是可变的nConstitutive alte

28、rnative splicing: more than one product is always made from a pre-mRNAnRegulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue typesAn example of constitutive alternative splicing : Splicing of the SV40 T ant

29、igen RNAFigure 13-16选择性剪接的调节通过激活子和抑制选择性剪接的调节通过激活子和抑制子实现子实现 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 SR

30、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.hnRNPs binds RNA and act as repressors

31、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.Regulated alternative splici

32、ngFigure 13-17Binds at each end of the exon and conceals (隐藏) it Coats the RNA and makes the exons invisible to the splicing machineryAn example of repressors: inhibition of splicing by hnRNPIFigure 13-18选择选择性剪接的性剪接的结结果果:1. Producing multiple protein products, called isoforms.2. Switching on and off

33、 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. 有一小类的内含子的剪接通过小剪接有一小类的内含子的剪接通过小剪接体实现体实现 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.Figure 13-19The AU-AC spli