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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
2、倍。,3.7.2 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 D The 5 end of tRNA is generated by a cleavage action catalyzed by the enzyme RNase 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 transfe
3、rase)催化下进行，由CTP和ATP提供胞苷酸和腺苷酸,原核生物,3.7.3 原核生物rRNA的加工,Processing of pre-rRNA transcripts in bacteria. 1 Before 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
4、RNase III, RNase P, and RNase E, respectively. RNase P is a ribozyme. 3 The final 16S, 23S, and 5S rRNA products 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 includ
5、ed in the primary transcript. Some copies of the gene have additional tRNA gene segments between the 16S and 23S rRNA segments and at the far 3 end of the primary transcript.,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(
6、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.,
7、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
8、上，或者虽然位于内含子中间但干扰了前体mRNA的正常剪接。,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，
9、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-protein 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=UAGAAGGAG,The 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 the
10、polypyrimidine (Py) tract/3 splice site,snRNA pairing is important in splicing,U1 snRNP initiates splicing,以碱基互补的方式识别pre-mRNA的5剪接点,U2 pairs with the branch site,U6 pairs with the 5-splicing site,西北农林科技大学 郭泽坤,U2AF (U2 auxiliary factor), recognizes the polypyrimidine (Py) tract/3 splice site,早期复合体E,A
11、复合体,B1 复合体,B2 复合体,C 复合体,U4的释放，促进U6和U2配对,U5 snRNP helps to bring the two exons together,U2, U5, U6 bring the reaction group close,一个矛盾（paradox）,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
12、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?,外显子遗漏,The first guard mechanism,This co-transcriptional loading process greatly diminishes the likelihood of exon skipping.,The second guard mechanism,SR (Serin
13、e 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 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 site,how does alternative
14、 splicing occur so often? The basic answer is that some splice sites are used only some of the time, leading to the production of different 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
15、 product is always made from the transcribed gene. In the case of regulated splicing, different forms are generated at different times, under different conditions, or in different cell or tissue types.,正常情况,外显子遗漏,外显子延伸,内含子保留,可变剪接,肌钙蛋白T,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 abu
16、ndant, this protein directs the machinery to favor use of the closest 5 splice site.,Alternative Splicing Is Regulated by Activators and Repressors,ESE: exonic splicing enhancer ISE: intronic splicing enhancer ESS: exonic splicing silencer ISS: intronic splicing silencer,Most of activators are recognized by SR protein The SR protein family-which is large and diverse-has specific roles in regulated alternative splicing as well, by directing the splicing machine
