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RNA的转录后加工(1)分子生物学_图文

3.7 真核生物RNA的转录后加工
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)

3.7.1 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8~10个内含子,前体分子一般比成熟mRNA大4~ 10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 transferase)催化下进行,由CTP和ATP提供胞苷酸 和腺苷酸

原核生物

真核生物tRNA前体的加工
?
? ?

? ?

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

3.7.3 原核生物rRNA的加工

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 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 included 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.
Processing of pre-rRNA transcripts in bacteria.

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

3.7.4 真核生物mRNA前体的加工
? 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)

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)

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。

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

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,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=UAG>AAG>GAG

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 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premRNA的5’剪接点

U2 pairs with the branch site

U6 pairs with the 5?-splicing site

早期复合体E

A 复合体

B1 复合体

B2 复合体

C 复合体
U2AF (U2 auxiliary factor), recognizes the polypyrimidine (Py) tract/3' splice site, 西北农林科技大学 郭泽坤

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 5’splice site may be able to react with any 3’splice site. But in the usual circumstances splicing occurs only between the 5?and 3?sites of the same intron.
What rules ensure that recognition of splice sites is restricted so that only the 5’and 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 (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 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 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 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 abundant, 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 machinery to different splice sites under different conditions ? Each 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 splicing 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。

? ?

Most 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 splicing machinery. Instead, by blocking specific splice sites, they repress the use of those sites.

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。

RNA剪接的调控

某些可变剪接的外显子总是在成熟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。

5. I类和II类自剪接内含子
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。 生物体内各种内含子
内含子类型 细胞内定位 细胞核,pre-mRNA(真核) 细胞核,pre-mRNA(真核) 细胞核,pre-mRNA(真核),细胞器RNA,少数细菌RNA 细胞器RNA,部分细菌RNA 细胞器RNA 细胞器RNA 细胞核,pre-tRNA(真核)

GU-AG
AU-AC I类内含子

II类内含子
III类内含子 双内含子 Pre-tRNA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 in 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, and

a

guanine nucleotide cofactor.
No other base can be substituted for G; but a triphosphate is not needed; 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 number of proteins。 The proteins help stabilize the correct structure-partly by shielding屏蔽 the negative charges provided by the phosphates in those backbone regions

RNA通过折叠形 成一个鸟苷结合 口袋,结合游离 的G

L19-RNA同样具有催化活性
The L-19 RNA is generated by opening the circular intron
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
Figure 26.8 illustrates the mechanism by which 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 3’–terminal G, and then back to a new C5 molecule. Further transfer reactions 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.

Group I introns form a characteristic secondary structure
?Group 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 G
IGS: internal guide sequence. 内部引导序列

酶活性和底物的多样性
序列特异性内切核酶 类似于第一步转酯反应

RNA连接酶

磷酸酯酶

核 酶 具 有 多 种 催 化 活 性

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 folds 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 (provided by an internal 2? –OH position in group II introns). In the second reaction, the free 3? –OH at the 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

II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 A mRNA 序列: GAU UGU AUA 蛋白质 序列: Asp Cys Ile

位点特异性脱氨基作用

site-specific deamination

人载脂蛋白B基因

位点特异性脱氨基作用

site-specific deamination
? 载脂蛋白C→U导致提前终止 ? 由脱氨基酶催化

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的编辑不是很普遍。

RNA editing can be directed by guide RNAs 指导RNA
?

A 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 or 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.

?

Editing 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 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-edited mRNA. The substrate RNA is cleaved at a site that is (presumably) identified by the absence of pairing with the guide RNA, a uridine is inserted or deleted to base pair with the guide RNA, and then the substrate RNA is ligated.

?

?

?

UTP provides the source for the uridyl residue. It is added by the TUTase activity;
it is not clear whether this activity, or a separate exonuclease, is responsible for deletion.

?

西北农林科技大学 郭泽坤

RNA的编辑的生物学意义:
? 校正作用 有些基因在突变过程中丢失的遗传

信息可能通过RNA编辑修复。 ? 调控翻译 通过编辑可以构建和去除起始或终

止密码子,是基因表达调控的一种方式。 ? 扩充遗传信息 能是基因产物获得新的结构

和功能,有利于生物进化。

3.8.2 RNA的再编码
mRNA在某些情况下不是以固定的方式被翻译,

而可以改变原来的信息,以不同的方式进行翻译,
科学上把RNA编码和读码方式的改变称为RNA的再 编码(RNA recoding)。
RNA的再编码的表现方式: 1. 2. 3. 核糖体程序性+1/-1移位 核糖体跳跃 终止子通读(硒代半胱氨酸、吡咯赖氨酸)

再编码可以从一个mRNA产生两种或多种相互关联但又不同的蛋白质,这 也可能是蛋白质合成的一种调节机制。

3.8.3 RNA的化学修饰

snoRNA参与了RNA的化学修饰

3.8.4 核酶
? 核酶(ribozyme)是指一类具有催化功能的RNA 分子,通过催化靶位点RNA链中的磷酸二酯键的

断裂,特异性地剪切底物RNA分子,从而阻断基
因的表达。 ? 核酶的催化功能与其空间结构有密切关系,具有 自我剪切能力的RNA大多数都能形成锤头结构。

催化分子内反应

催化分子间反应

按核酶的作用方式可分为剪切型(把RNA前体的多余部分切除),和剪接型(

把RNA前体的内含子部分切除并把不连续的外显子部分连接起来)。

剪切型:只剪不接,催化自身或不同RNA分子,切下特异的核苷酸序列。 剪接型:具有序列特异的内切核酸酶、RNA连接酶等多种酶的活性。既能切割 RNA分子,也能通过形成新的磷酸二酯键连接切割后的RNA分子。

锤头型核酶和发夹型核酶都能催化产物的连接
,但发夹型核酶催化连接反应的活性比切割反 应的活性高10倍,而锤头型核酶催化切割反应 的活性比连接反应的活性高100倍。

核酶发现的意义:

3.8.5 RNA在生物进化中的地位
酶组成的进化:

生物体组成的进化:


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