RNA的转录后加工分子生物学

1、1977年Robert J and Sharp P A分别发现断裂基因（interrupted gene），1993年获得诺贝尔生理学和医学奖当用RNA与其转录的模板DNA分子杂交时，RNA链取代DNA双链中对应的链，形成R-突环(R-loop)3.7 真核生物RNA的转录后加工3.7.1 RNA中的内含子真核生物的基因往往是断裂的基因，其转录所形成的RNA前体要经过剪切，将内含子切除后，将外显子拼接起来才能形成成熟的mRNA。存在于真核生物基因中无编码意义而被切除的序列。在前体RNA中的内含子也常被称作“间插序列”。 真核基因平均含810个内含子，前体分子一般比成熟mRNA大410倍。3.7

2、.2 tRNA前体的加工加工tRNA前体3端的核酸内切酶是RNase F负责修剪的核酸外切酶可能主要是RNase DThe 5 end of tRNA is generated by a cleavage action catalyzed by the enzymeRNase P细菌的tRNA前体存在两类不同的3端序列。一类其自身具有CCA三核苷酸，位于成熟tRNA序列与3端附加序列之间，当附加序列被切除后即显露出该末端结构，另一类其自身并无CCA序列，当前体切除3端附加序列后，必须外加CCA。添加CCA是在tRNA 核苷酰转移酶(nucleotidyl transferase)催化下进行，由

3、CTP和ATP提供胞苷酸和腺苷酸原核生物真核生物tRNA基因的数目比原核生物tRNA基因的数目要大得多。真核生物的tRNA也成簇排列，并且被间隔区所分开真核生物tRNA前体的3端不含CCA序列，成熟tRNA 3端的CCA是后加上去的，由核苷酸转移酶催化此反应。tRNA的修饰成分由特异的修饰酶所催化。真核生物的tRNA除含有修饰碱基外，还有2-O-甲基核糖，其含量约为核苷酸的百分之一。有些tRNA还具有居间序列，需要进一步拼接真核生物tRNA前体的加工3.7.3 原核生物rRNA的加工Processing of pre-rRNA transcripts in bacteria. 1 Before

4、 cleavage,the 30S RNA precursor is methylated at specific bases. 2 Cleavage liberates precursors of rRNAs and tRNA(s). Cleavage at the points labeled 1, 2, and 3 is carried out by the enzymes RNase III, RNase P, and RNase E, respectively. RNase P is a ribozyme. 3 The final 16S, 23S, and 5S rRNA prod

5、ucts result from the action of a variety of specific nucleases. The seven copies of the gene for pre-rRNA in the E. coli chromosome differ in the number, location, and identity of tRNAs included in the primary transcript. Some copies of the gene have additional tRNA gene segments between the 16S and

6、 23S rRNA segments and at the far 3 end of the primary transcript.6真核生物rRNA前体的加工哺乳动物转录产生45S rRNA前体。果蝇是38S，酵母是37S在核仁内进行RNA的拼接共有4种方式：核mRNA的拼接体的拼接(nuclear mRNA spliceosomal splicing)类型I自我拼接(group I self-splicing)类型II自我拼接(group II self-splicing)核tRNA的酶促拼接（nuclear tRNA enzymatic splicing）3.7.4 真核生物mRNA前体

7、的加工细胞内已发现的RNA剪接有3种（不包括tRNA的加工）。hnRNA的拼接GT-AG原则（GT-AG rule, GU-AG rule）此规则不适合于线粒体和叶绿体基因的内含子，也不适合于tRNA和rRNA的内含子The branch site lies 18-40 nucleotides upstream of the 3 splice site.GU-AG指的是内含子的两端序列在内含子内部部分序列也可能参与内含子的剪接。他们可能是pre-mRNA剪接过程中各种核糖核蛋白剪接调节因子的结合位点，对于有效和准确的剪接非常重要。剪接的普遍性任何单个mRNA前体的剪接点是通用的，无特异性；剪接

8、装置无组织特异性，一个RNA 分子在任何细胞均可被正确地剪接。剪接的特异性比较同源基因的进化过程发现内含子的异化大于外显子，特定的内含子还可能在进化过程中丢失，因此内含子的功能及其在生物进化中的地位是一个引人注目的问题，另外，许多人类疾病是内含子剪接异常引起的，如地中海贫血患者的珠蛋白基因中，大约有1/4的核苷酸图标发生在内含子的边界保守序列上，或者虽然位于内含子中间但干扰了前体mRNA的正常剪接。13 pre-mRNA剪接的机制和套索结构第一阶段，内含子的5端切开，左侧的外显子呈线状，右侧的内含子-外显子分子形成一个套索(Lariat)结构。内含子游离的5端通过5-2磷酸二酯键与分支位点的A

9、相连。第二阶段，内含子的3剪接点被切断然后以套索状释放，与此同时两侧的外显子连在一起。 两阶段同时发生。腺苷酸原来已有3,5-磷酸二酯键依然存在，加上此2,5-磷酸二酯键连接后，在腺苷酸处出现了一个套索。RNA 剪接由剪接体（splicesome）执行转酯反应是由一个称为剪接体的大复合体介导的。剪接体中包含约150种蛋白和5种小RNA，大小类似核糖体。RNA 和蛋白质都参与共有序列的识别。剪接体的多数功能是由RNA分子执行的； RNA分子识别内含子与外显子交界序列，亲自催化剪接。5 种核内小RNA（ snRNA） ：U1，U2，U4，U5，U6 （序列中富含U）与蛋白质形成 RNA-prote

10、in 复合物称为小的核内核糖核蛋白 (snRNP）。 AG前一位核苷酸可以影响剪切效率：CAG=UAGAAGGAGThe snRNPs 在剪接中有3个功能：识别5 剪接位点和分支点; 把这2个位点集结到一起; 催化或协助催化剪接和连接反应。U2AF (U2 auxiliary factor), recognizes thepolypyrimidine (Py) tract/3 splice site,snRNA pairing is important in splicingU1 snRNP initiates splicing以碱基互补的方式识别pre-mRNA的5剪接点20U2 pairs

11、 with the branch site21U6 pairs with the 5-splicing site22 西北农林科技大学 郭泽坤U2AF (U2 auxiliary factor), recognizes thepolypyrimidine (Py) tract/3 splice site,早期复合体EA 复合体B1 复合体B2 复合体C 复合体23U4的释放，促进U6和U2配对24U5 snRNP helps to bring the two exons together25U2, U5, U6 bring the reaction group close 26一个矛盾（par

12、adox）In principle any 5splice site may be able to react with any 3splice site. But in the usual circumstances splicing occurs only between the 5and 3sites of the same intron. What rules ensure that recognition of splice sites is restricted so that only the 5and 3 sites of the same intron are spliced

13、?27外显子遗漏28The first guard mechanismThis co-transcriptional loading process greatly diminishes the likelihood of exon skipping.29The second guard mechanism SR (Serine Argenine rich) proteins bind to sequences called exonic splicing enhancers (外显子剪接增强子ESEs) within the exons. SR proteins bound to these

14、 sites interact with components of the splicing machinery SR proteins recruit the U2AF proteins to the 3 splice site and U1 snRNP to the 5 site3031how does alternative splicing occur so often? The basic answer is that some splice sites are used only some of the time, leading to the production of dif

15、ferent versions of the RNA from different transcripts of the same gene. Alternative splicing can be either constitutive or regulated. In the former case, more than one product is always made from the transcribed gene.In the case of regulated splicing, different forms are generated at different times

16、, under different conditions, or in different cell or tissue types.32正常情况外显子遗漏外显子延伸内含子保留可变剪接33肌钙蛋白T34两种抗原的比例因剪接相关蛋白SF2/ASF的表达水平而不同。SF2/ASF is an SR protein, when abundant, this protein directs the machinery to favor use of the closest 5 splice site.35Alternative Splicing Is Regulated by Activators a

17、nd RepressorsESE: exonic splicing enhancerISE: intronic splicing enhancerESS: exonic splicing silencerISS: intronic splicing silencer36Most of activators are recognized by SR proteinThe SR protein family-which is large and diverse-has specific roles in regulated alternative splicing as well, by dire

18、cting the splicing machinery to different splice sites under different conditionsEach SR protein has another domain, rich in arginine and serine, called an RS domain.The RS domain, found at the C-terminal end of the protein, mediates interactions between the SR protein and proteins within the splici

19、ng machinery.SR蛋白家族数量庞大种类多样，在不同的条件下引导剪接体到不同的剪接位点发挥作用。在发育的某个阶段，或者在某种类型的细胞中，一种特定的SR蛋白的存在与否或者活性高低，就可以决定某一特定的剪接位点是否得到利用。37Most silencers are recognized by members of the heterogeneous nuclear ribonucleoprotein (核不均一核糖核蛋白hnRNP) family. These bind RNA but lack the RS domains and so cannot recruit the spli

20、cing machinery. Instead, by blocking specific splice sites, they repress the use of those sites.38有时hnRNP1结合到多聚嘧啶区，全面阻断剪接体的结合；有时它结合到某个外显子两外侧的序列上，使该外显子不能进入成熟的mRNA。单个外显子的剔除有两种可能的原因：外显子两端的hnRNPI通过相互作用，使外显子突起成一个环，当剪接体经过时把它漏掉。或者该外显子两端的hnRNPI与其他hnRNPI协同结合，将外显子所处的一段RNA覆盖起来，使得剪接体“看不到”这个外显子。39RNA剪接的调控某些可变剪接的

21、外显子总是在成熟mRNA出现，除非受到某种抑制蛋白的阻止（见a),另外一些则相反，只有某种激活因子发挥作用，才能包含在成熟的mRNA中（见b)。这种剪接调控使得某一特定的外显子出现在一种类型细胞的成熟mRNA中，而不会出现在另一种类型细胞的成熟mRNA中。与mRNA前体中主要（GU-AG类）和次要（AU-AC类）内含子剪接方式不同，I、II类内含子能进行自我剪接。内含子类型细胞内定位GU-AG细胞核，pre-mRNA（真核）AU-AC细胞核，pre-mRNA（真核）I类内含子细胞核，pre-mRNA（真核），细胞器RNA，少数细菌RNAII类内含子细胞器RNA，部分细菌RNAIII类内含子细胞

22、器RNA双内含子细胞器RNAPre-tRNA内含子细胞核，pre-tRNA（真核）生物体内各种内含子5. I类和II类自剪接内含子类型I自我拼接Autosplicing (Self-splicing) describes the ability of an intron to excise itself from an RNA by a catalytic action that depends only on the sequence of RNA in the intron.Group I and group II introns are found in organelles and i

23、n bacteria. (Group I introns are found also in the nucleus in lower eukaryotes.) Group I and group II introns are classified according to their internal organization.Each can be folded into a typical type of secondary structure.The reaction requires only a monovalent cation（单价阳离子）, a divalent cation

24、, and a guanine nucleotide cofactor. No other base can be substituted for G; but a triphosphate is notneeded; GTP, GDP, GMP, and guanosine itself all can be used, so there is no net energy requirement. The guanine nucleotide must have a 3 OH group In vivo, self-splicing intron is complexed with a nu

25、mber of proteins。The proteins help stabilize the correct structure-partly by shielding屏蔽 the negative charges provided by the phosphates in those backbone regionsRNA通过折叠形成一个鸟苷结合口袋，结合游离的GL19-RNA同样具有催化活性The L-19 RNA is generated by opening the circular intron Figure 26.8 illustrates the mechanism by w

26、hich the oligonucleotide C5 is extended to generate a C6 chain. The C5 oligonucleotide binds in the substrate-binding site, while G414 occupies the G-binding site. By transesterification reactions, a C is transferred from C5 to the 3terminal G, and then back to a new C5molecule. Further transfer rea

27、ctions lead to the accumulation of longer cytosine oligonucleotides. The reaction is a true catalysis, because the L-19 RNA remains unchanged, and is available to catalyze multiple cycles.线型L-19 RNA可催化寡聚胞苷酸（C5）延长，使两个C5转化为一个C4和一个C6，称为polyC聚合酶活性或核苷酸转移酶活性Group I introns form a characteristicsecondary s

28、tructureGroup I introns form a secondary structure with 9 duplex regions.The core of regions P3, P4, P6, P7 has catalytic activity.Regions P4 and P7 are both formed by pairing between conserved consensus sequences.A sequence adjacent to P7 base pairs with the sequence that contains the reactive GIGS

29、: internal guide sequence.内部引导序列酶活性和底物的多样性核酶具有多种催化活性序列特异性内切核酶RNA连接酶磷酸酯酶类似于第一步转酯反应类型II自我拼接Group II introns excise themselves from RNA by an autocatalytic splicing event.The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns.A group II intron fold

30、s into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron.In the first reaction, the 5 exon-intron junction is attacked by a free hydroxyl group (providedby an internal 2 OH position in group II introns). In the second reaction, the free 3 OH at th

31、e end of the released exon in turn attacks the 3 intron-exon junction主要存在于真核生物的线粒体和叶绿体rRNA基因中。完成第一次转酯反应的催化部位，其结构在II类自剪接内含子和pre-mRNA/snRNP复合体中高度相似 西北农林科技大学 郭泽坤54II类自剪接的化学过程与剪接体介导的剪接反应过程基本相同，由内含子内高度活泼的腺苷酸启动剪接过程，并形成套索状产物。I类自剪接内含子的RNA通过折叠形成鸟苷结合口袋，从而结合一个游离的鸟苷用以启动剪接过程。虽然这类内含子在体外无需蛋白质协助就能够进行自身剪接，但在体内环境下，它们

32、通常需要蛋白质组分来激活剪接反应。3.8 RNA的编辑、再编码及化学修饰3.8.1 RNA的编辑 RNA的编辑是某些RNA，特别是mRNA前体的一种加工方式，如插入、删除或取代一些核苷酸残基，导致DNA编码的遗传信息的改变。 介导RNA编辑的机制有两种： 位点特异性脱氨基作用； 引导RNA指导的尿嘧啶插入或删除。DNA正链序列： GA G A AmRNA 序列： GAU UGU AUA蛋白质 序列： Asp Cys Ile人载脂蛋白B基因位点特异性脱氨基作用site-specific deamination 位点特异性脱氨基作用site-specific deamination 载脂蛋白CU导

33、致提前终止由脱氨基酶催化RNA的编辑不是很普遍。脱氨基是由脱氨酶催化，在RNA编辑时脱氨酶亚基复合体能识别特异性靶位点。RNA editing can be directed by guide RNAs指导RNAA guide RNA is a small RNA whose sequence is complementary to the sequence of an RNA that has been edited. It is used as a template for changing the sequence of the pre-edited RNA by inserting o

34、r deleting nucleotides.Extensive RNA editing in trypanosome（锥虫） mitochondria occurs by insertions or deletions of uridine.The substrate RNA base pairs with a guide RNA on both sides of the region to be edited.The guide RNA provides the template for addition (or less often deletion) of uridines.Editi

35、ng is catalyzed by a complex of endonuclease, terminal uridyltransferase （尿苷酰转移酶）activity, and RNA ligase.In 1986, Rob Benne and his colleagues discovered that the sequence of the cytochrome oxidase(细胞色素氧化酶COXII) mRNA from trypanosomes does not match the sequence of the COII gene.Part of the edited

36、sequence of the COXIII mRNA of T.Brucei布鲁氏菌尿苷酸的缺失和添加a model for gRNA action in the cytochrome b gene of Leishmania（利什曼虫）Editing of uridines is catalyzed by a 20S enzyme complex that contains an endonuclease, a terminal uridyltransferase (TUTase), and an RNA ligase。It binds the guide RNA and uses it to pair with the pre-ed