1、Chapter 7 RNA SplicingChapter 13 of the bookIn 1977,Robert J and Sharp P A discovered interrupted genes individually.A typical eukaryotic geneRNA 拼接拼接（RNA splicing）：introns are removed from the per-mRNA by a process called RNA splicing.Some pre-mRNAs can be spliced in more than one way,generating al
2、ternative mRNAs.This is called alternative splicing.拼接点：5 拼接点或左拼接点（内含子上游）3 拼接点或右拼接点（下游）类内含子（group）:含有中部核心结构的 细胞器基因 核基因 类内含子（group）:不含有中部核心结构 细胞器线粒体基因内 核基因 GUAG内含子（group ）:具有 GUAG 特征 的边界序列 核基因mRNA前体 tRNA基因的内含子 均位于 tRNA 的反密码环上 Introns四种内含子的边界序列各有一定共同的特征3、拼接方式 Class I：由拼接复合物完成（核mRNA内含子）可供识别的特异序列 拼接复合物由
3、多种蛋白质和snRNA组成 Class II：自我拼接（Group、）形成特定的二级结构 RNA具有催化拼接的能力 Class III：需要蛋白质酶参与的拼接（酵母tRNA）前两种拼接都属于转酯反应splicing of tRNA in yeast 1、链的断裂和连接是两个独立的过程 2、tRNA的内含子均位于反密码环的3端，与反密码 子相距 一个nt 长1416bp，其中含一段与反密码环互补的序列反密码环处形成一个与成熟tRNA不同的构象 只有anti-code受到影响酵母tRNAphe 第一步：内切酶作用 释放一条线状内元分子和两个“tRNA半分子”两个tRNA半分子采取成熟tRNA分子的
4、构象(切刻)拼接过程 第二步：RNA连接酶连接断端 其中内切酶作用后产生 5OH 和 3磷酸，3磷酸端很快转 变为2，3环式核苷酸 因此，一个半分子有两个磷酸末端，一 个半分子有两个 OH末端连接反应前要进行两个反应：a、左外元的3端成为OH （环式磷酸二酯酶）其 3 位为OH，2 位为磷酸b、第二个外元的 5端转变为 磷酸基（多聚核苷酸激酶）因此连接后还要切除第一个外元的 2磷酸这是 tRNA内元拼接重要特点之一Splicing of Classintron（self-splicing）1、结构特点：（1）5 拼接点和3 拼接点-U G 5 exonUintronGexon3 自我拼接分为；
5、两种方式：I类内含子：梨形四膜虫 II类内含子：真核生物的线粒体和叶绿体rRNA基因 The mechanism of splicing in class I intron（四（四膜虫的大膜虫的大rRNA前体的前体的拼接）拼接）Step 1：游离G发动的转酯反应 G 的 3-OH 攻击内含子的 5拼接点 G-内含子；左外显子（有游离3OH）Step 2：游离外显子（左）发动的转酯反应 左外元的3-OH 攻击3拼接点，同时释放 线状的内含子,形成成熟RNA分子 两次转酯反应是紧密偶联的 释放出的内元可继续进行转酯反应 而形成环状 自我拼接过程主要由转酯反应构成，且完全由内元自身 完成-自我拼接的
6、最大特点 转酯反应是将一处已有的磷酸二酯键变成另一处新的 磷酸二酯键 内元自身能形成特定的二级结构，产生适于拼接反 应的构象和活性位点,在G的参与下完成拼接反应 (内部引导序列)科罗拉多大学Cech等人完成（美）活性位点 这一机制如下图所示:四膜虫的 35SRNA 的内含子有自身催化的性质 即将一处已有的磷酸二酯键转变成另一处新的磷 酸二酯键，不需要额外的能量 因此：内含子可以看作是 RNA重排酶（RNA rearrangease）或 RNA异构酶（RNA isomerase）其中：拼接反应中，“酶”和底物是同一个分子，且反应后 变成了不同的分子RibozymeA ribozyme is an
7、 RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction.拼接点序列 Breathnach-Chambon rule（GUAG规则）5-exon-GU-intron-AG-exon-3供点 100%94%受点 7nt branch site 3拼接点的上游 py X py U pu A pySplicing of class II introns (splicesome)The snRNPs involved in splicing,together
8、with many additional proteins,form a large particulate complex,called the spliceosome.RNA Splicing is carried out by spliceosom.snRNA(小核内RNA)：U1，U2，U4，U5，U6snRNP,(小核内核糖核蛋白)：snRNA-protein complexes are called small nuclear ribonuclear proteins.lariat Splicing mechanismSplicing mechanism of class II i
9、ntronsof class II introns SnRNA(or ScRNA)与拼接点序列间存在互补区域 并参与剪接,形成拼接体(spliceosome)。Spliceosome 逐级组装，SnRNA（U1、U2、U5和 U4U6）分步替代 U1通过与 5拼接点互补而结合 U2识别并结合分支点A U1和U2作用使内元的 5端和 3端带到 一起（U1与 3拼接点配对）U1、U2、mRNA与U4U5U6复合物形成一个完整的拼接体 第一次转酯左外元、内元剪切套索lariat 第二次转酯exons连接、套索状内元释放 拼接体（spliceosome）解体与lariat降解同步反式剪接的情况较为稀少
10、，较典型的例子是锥虫表面糖蛋白基因VSG(variable surface glycoprotein)，线虫的肌动蛋白基因（actin genes），和衣藻（chlamydomonas）叶绿体DNA中含有的psa基因。Trans-splicing 锥虫的许多mRNA都含有这个 35bases 序列,称其为前导序列（leader sequence).前导序列是其基因中所没有的.这种前导序列来源于基因组中位于别处的重复序列（200个）所转录的小片段RNA，每个重复单位长135nt，前面35nt的前导序列可以通过转酯反应加到 mRNA的5端。splice leader(SL)Alternative
11、splicing An example of alternative splicingAlternative splicing of SV40 T-antigenThe possible mechanisms of alternative splicing1.Major and minor splicesome2.nonsense-mediated decay3.A novel strategy used by Dscam Exon 6 Drosophila Dscam gene-encodes cell surface proteins of the Ig superfamilyAltern
12、ative splicing is regulated by activators and repressorsRegulation of alternative splicing determine the sex of the fliesExon shufflingthe significance of broken genes in eukaryotes?Alternative splicing Reshuffling exonsThree evidences1.The borders between exons and introns within a given gene often
13、 coincide with the boundaries between domains within the protein encoded by that gene.2.Many genes have apparently arisen during evolution in part via exon duplication and divergence.3.Related exons are sometimes found in otherwise unrelated genes.There are numerous examples of proteins made up of h
14、ighly related domains used in various combinations,encoded by genes made up of shuffled exons.RNA编辑（RNA editing）RNA editing is a process in which information changes at the level of mRNA.e.g.DNA正链序列：GA G A A mRNA 序列：GAU UGU AUA 蛋白质 序列：Asp Cys IleIn 1986,Rob Benne and his colleagues discovered that t
15、he sequence of the cytochrome oxidase(COII)mRNA from trypanosomes does not match the sequence of the COII gene.RNA editing occurs in two different situations,with different causes.2.The mechanism of the editosome involves an endonucleolytic cut at the mismatch point between the guide RNA and the une
16、dited transcript.The next step is catalyzed by one of the enzymes in the complex,a terminal U-transferase,which adds Us from UTP at the 3 end of the mRNA.The opened ends are held in place by other proteins in the complex.Another enzyme,a U-specific exoribonuclease,removes the unpaired Us.After editi
17、ng has made mRNA complementary to gRNA,an RNA ligase rejoins the ends of the edited mRNA transcript.Functions of RNA edit 校正作用调控翻译扩充遗传信息mRNA transportMany proteins are involved in this process.Mature mRNA carries a collection of proteins that identifies it as being mRNA destined for transport.Other RNAs not only lack the particular signature collection of proteins for transport,but have their own alternative sets of proteins that actively block export.Once a RNA has been fully processed-capped,spliced,and polyadenylated-an mRNA is transported out of the nucleus and into the cytoplasm
