RNA Posttranscriptional
Procession
转录后加工
Classes of RNA
Pre-RNA,前体 RNA,The primary transcript has the same
organization as the gene
hnRNA,heterogeneous nuclear RNA,核内不均一 RNA,Eucaryotic
RNA Pol II transcribes a variety of different genes
snRNA,small nuclear RNA,核内小 RNA,is any one of many small
RNA species confined to the nucleus; several of the snRNAs are
involved in splicing or other RNA processing reactions
scRNA,small cytoplasmic RNA,胞内小 RNA,several molecules
present in the cytoplasm and (sometimes) nucleus
rRNA,ribosomal RNA,核糖体 RNA,Serve as components of
ribosomes
tRNA,transfer RNA,转移 RNA,carrying an amino acid; the
covalent linkage is between the NH2 group of the amino acid and
either the 3′- or 2′-OH group of the terminal base of the tRNA t
mRNA,messager RNA,信使 RNA,carries genetic message from
the chromosome to the ribosomes
Function of RNA
Class Function Position
hnRNA 成熟 mRNA的前体 核内
snRNA 参与 hnRNA的剪接、转运 核内
scRNA 蛋白质内质网定位合成的信 号识别体的组成成分 胞内
rRNA 核蛋白体组成成分
tRNA 转运氨基酸
mRNA 蛋白质合成模板
Types of RNA processing
A) Cutting and trimming to generate ends:
– rRNA,tRNA and mRNA
B) Covalent modification:
– Add a cap and a polyA tail to mRNA
– Add a methyl group to 2’-OH of ribose in mRNA and
rRNA
– Extensive changes of bases in tRNA
C) Splicing
– pre-rRNA,pre-mRNA,pre-tRNA by different
mechanisms.
The Long and Winding
Road…..
Eukaryotic and Prokaryotic mRNAs
Have Major Differences - I
Eukaryotic and Prokaryotic mRNAs
Have Major Differences - II
Ribosome核糖体
A supramolecular complex
of rRNAs and proteins,the
site of protein synthesis
Have two subunit
In eukaryotes,binding in
RER; in prokaryotes,
binding in mRNA
一,rRNA processing in
prokaryotes
Seven different operons for rRNA rrn dispersed
thought out the genome
Each operons transcript a 30S(~6000bp) primary
transcript
Each primary transcript contains one copy of
5S,16S and 23S rRNA,together with some tRNA
sequence
Rnase III is involved in rRNA processing
Rnase in E.coli
E.coli中含有的小分子核酸酶酶 基因 底物 功能
RNaseP rnpA tRNA 5′ 端 内切
RnaseO rnpB
RnaseBN? tRNA 3′ 端 外切
RnaseD rnd tRNA 3′ 端 外切
RnaseT? tRNA 3′CCA 外切
RnaseⅢ rnc rRNA和 mRNA 内切
RnaseR? rRNA和 mRNA 外切
RnaseE rne 5S rRNA 内切
RnaseⅠ rna 大部分 RNA 内切
RnaseⅡ rnb RNA 外切多核苷酸磷酸化酶 pnp RNA 外切
RnaseH rnh RNA-DNA杂合链 内切
The primary transcript
Ribonucleoprotein 核糖核酸蛋白复合物 RNP complex
The precursor rRNA folds up
into stem-loop structures by
base pairing in the transcript
Some specific proteins bind to
the rRNA to form RNP complex
Steps in rRNA processing
rRNA前体被大肠杆菌 RNaseⅢ,RNaseE等剪切成一定链长的 rRNA分子
rRNA在修饰酶催化下进行碱基修饰
rRNA与蛋白质结合形成核糖体的大、小亚基
Pre-16S rRNA Pre-5S rRNAPre-t RNA Pre-23S rRNA Pre-tRNA
Precursors
Mature rRNA
Primary
transcript
RNase
Primary rRNA processing in prokaryotes
Eukaryotic rRNA gene organization
In the nucleolus,RNA polymerase I transcribes the
rRNA,which usually exist in tandem repeats
containing 100 or more clusters of the transcription
units
A nucleolus is formed around each cluster
RNA polymerase III transcribes 5S rRNA genes,not in
cluster with other rRNA genes its gene copy number
do not coordinate with other RNA genes;
Primary transcript 47S,containing
– ETS external transcribed sequence外部转录间隔区
– ITS internal transcribed sequence内部转录间隔区
– NTS,non-transcribed spacer非转录间隔区
Some rRNA genes have intron
Eukaryotic rRNA gene organization
rRNA is synthesized in the nucleolus and
assembled with proteins to form subunit
the subunit is then transported into cytoplasm
rRNA processing in eukaryotes
The precursor folds and complexes with proteins
RNP as it is being transcribed in the nucleolus
Many specific ribose methylations take place at
over 100 sites and is carried out directly by a
subset of snRNP particles
snRNPs contain snRNAs that have short streches
of complementarity to parts of the rRNA and,by
base pairing with it,they define where methylation
takes place
pre-rRNA processing is accompanied by the
formation of 60S and 40S subunits
Processing of pre-rRNA
Cleavage is by
ribonucleases,核糖核酸酶,RNA酶
5.8S rRNA is H-
bonded to 28S rRNA
in mature ribosome
Introns in pre-rRNA gene
Only a few organisms’ rRNA gene has introns
Splicing剪接 does not need protein,Ribozyme is
a catalytic RNA molecules that catalyze particular
biochemical reactions in the absence of protein
Introns that does not need proteins for splicing are
categorized as the group I intron
In mRNA,tRNA & rRNA genes in mitochondria
and chloroplasts of yeast/fungi,In rRNA genes of
all insects,In rRNA genes,In some mRNA and
tRNA genes of bacteriophage
Self-splicing自我剪接 for the group I
intron in trtrahymena pre-rRNA
Cleavage and ligation are
two steps here
Cleavage at the 5’ splice
junction,add G at the end
Cleavage at the 3’splice
juntion
Ligation of the exons
Cleavage of the lariat intron
Lariat is an intermediate in
RNA splicing in which a
circular structure with a tail
is created by a 5′-2′ bond
Self-splicing of
trtrahymena pre-rRNA
Need guanosine or
GNP’s 3’-OH
Three phosphoester
bond transfers
Concentration
independent
Not a real enzyme
二,3-D structure of tRNA
Post-transcriptional processing of pre-
tRNA
Addition of the 5’-CCA-3’ to the 3’ end
Chemical modification of the selected
bases,the type and extent of modification
varies
– Specific modification?3D-structure?
–?function (aa-specificity)
Removal of the extra sequences
– 5’-leader sequence,3’-trailer sequence
pre-tRNA
The 5’ and 3’ trailer
sequences are
removed by specific
enzyme during
processing
Processing of eukary,
Pre-tRNA is inside the
nucleus
Modified bases in tRNA
通过甲基化酶，硫醇酶，假尿嘧啶核苷化酶等进行修饰，
如氨基酸臂的 4-硫尿苷（ 4tu）,D臂的 2
甲基鸟苷（ 2mG），
TψC臂的假尿苷（ ψ）
和反密码子环上的 2
异戊腺苷（ 2ipA)
Prokaryote tRNA-genes
A cluster of tRNA genes may be transcribed
into one nascent transcript
5’-leader-(tRNA-spacer)n-tRNA-trailer-3’
Two known enzymes are involved in
processing,RNase P (5’-leader)
and RNase Q (3’-trailer)
Extra enzymes are needed for spacer removal
tRNA processing in prokaryotes
In prokaryotes,mature tRNA are generated
by processing longer pre-tRNA transcripts
by RNases D,E,F and P,followed by
base modification which are unique to each
particular tRNA type
Processing of pre-tRNA
Eukaryote tRNA-genes
真核 tRNA的基因和原核不同
– 真核的前体分子 tRNA是单顺反子，但成 簇排列，基因间有间隔区
– 真核 tRNA基因一般都比原核 tRNA基因多得多，如酵母约有 400个 tRNA基因
– 5′端单磷酸核苷酸，表明已被加工过
– tRNA的前体分子中含有内含子。
Processing of pre-tRNA
tRNA的加工分成 3个阶段
,斩头”，形成 5′末端
去尾，形成 3′-OH末端。缺 -CCA的 tRNA
要用 tRNA核酸转移酶 加 -CCA
修饰：通过甲基化酶，硫醇酶，假尿嘧啶核苷化酶等进行修饰，
4-thiouridine(S4U)4-硫尿苷； inosine(I)次黄嘌呤核苷，肌苷； 1-
methylguanosine(m1G)1-甲基鸟苷； N6-isopentenyladenosine (i6A) N6 -
异戊烯基腺苷； Ribothymidine(T)胸腺嘧啶核糖核苷；
Pseudouridine(ψ)假尿嘧啶核苷； dihydrouridine(D)二氢尿苷真核 tRNA内含子的特点：
位置相同，都在反密码子环的下游
不同 tRNA的内含子长度和序列各异
外显子和内含子交界处无保守序列
内含子的剪切是依靠 RNase异体催化
内含子和反密码子配对形成茎环
RNaseP 核糖核酸酶 P
Ribozyme核酶
RNaseP is endonuclease composed of one RNA
molecules and one protein molecules,It is a simple
RNP
RNaseP are found both in prokaryotes and eukaryotes
The RNA component alone works as an endonuclease,
This RNA is a catalytic RNA,or ribozyme
Ribozyme is a catalytic RNA molecules that catalyze
particular biochemical reactions in the absence of
protein
三,The processing of mRNA
hnRNA Eucaryotic RNA Pol II transcribes a variety of
different genes,from snRNA genes of 60-300nt to the large
Antennapedia genes
The hnRNA is rapidly covered in proteins to form hnRNP
The proteins involved have been classified as hnRNA proteins
A-U
These proteins are organized as a tetramer,Three copies of
tetramer are combined with a RNA molecule of 600-700nt,
called hnRNP particles,each particle has a size of 30-40S
The hnRNP protein are thought to help keep the hnRNA in a
single-stranded form and assist in the various RNA processing
reactions
These hnRNA are processed to give mRNAs called pre-mRNAs
snRNP Particles
snRNAs transcribed by RNA Pol II complex with specific
protein to form snRNP
These RNA are rich in uracil and are denoted as U1,U2 etc
U1,U2,U4,U5 and U6 are those involved in pre-mRNA
splicing,Many others seem to be involved in determining the
sites of methylation of pre-rRNA and are located in the
nulcleolus
The snRNA are synthesized in nucleus by RNA Pol II and
have a normal 5’-cap
They are exported to the cytoplasm where they associate with
the common core protein and other specific protein
Their 5’-cap gains two methyl groups and they are then
imported back into nucleus where they function in splicing
The procession of pre-mRNA
真核 mRNA的加工一般要经过四步：
5′加帽 (capping)
3′加尾 (tailing)
切除内含子 (intron cleavage )
修饰 (modification)：对某些碱基进行 甲基化
5’-capping
The 5’ end is modified by addition of m7G
residue,called a cap,The is nucleotide is added to
the new transcript in the reversed orientation,
giving 5’-5’ linkage
The sugars on the first and second transcribed
nucleotides are methylated,m7GNmpNmp
帽子结构的功能
(1)有助于 mRNA越过核膜，进入胞质；
(2)保护 5′不被酶降解；
(3)翻译时供 IFⅢ （ 起始因子 ） 和核糖体识别 。
加帽
(1) 剪接前加帽如呼肠病毒,牛豆病毒
(2) 剪切后加帽如疱疹病毒和口炎病毒剪接前加帽剪切后加帽帽子 的类型
在末端鸟苷的第 7位上存在单个甲基化位点的称 O型帽子 （ capO） ；
在次末端核苷酸的核糖上的 2′-0位点上还有一个甲基位点的称 1型帽子 (cap 1)；
此外,在第三个核苷酸的核糖上 （ 2′-0） 有甲基化位点的称 2型帽子 （ cap2）
这三种帽子都有特殊 面对面核苷酸结构
（ confrontde nucleotide structure）
三种帽子的共同 在帽 1 中可被甲基化 NH
2
位 置
C N
O C H
3
N C
C H
C N H C C
H N C N N
C H
O C O O O
2
HN
N
N
C H
2
O P O P O P O C H
O O O
帽 1 位 置 O CH
3
O
O P O C H
2
O
帽 2 位 置 O CH
3
O
O P O
O
图 1 3 - m R N A 5 ’ 端的帽子位置和可被甲基化的位点加尾 3’-tailing
成熟的 mRNA是前体 3‘端经过剪切再加上一串多聚 A即
poly(A)尾而形成的。
剪切和聚腺苷酸化需要 DNA和
pro-mRNA上的特定聚腺苷酸化序列 AAUAAA，在其后 11-
20nt处紧随一个 YA结构，在其下游常有一富含 GU的序列。
(1)特殊组分 (CPSF)识别
AAUAAA并指导其它的活性
(2) 剪切因子 (CF)在加尾位点
AAUAAA下游 11－ 30nt 处剪切
RNA；
(3)末端腺苷转移酶 (poly(A)聚合酶 PAP)合成 poly(A)尾巴；
(4) poly(A)结合 蛋白 (PBP)与
poly(A)结合，反应停止。
Polyadenylation signal
聚腺苷酸化信号
5’ Cap…..…..AAUAA..(20bases).CA………UUGUGUUG
signal Poly(A) GUrich region
cleavage site addition site
加 Poly(A)的反应第一步加一个短的寡聚 A序列
（ 10nt ）,此 反 应 依 赖 于
AAUAAA序列；
反应由 poly（ A） 聚合酶在特殊因子指导下完成的 。
第二步是 寡聚 A 尾巴延 伸到
240nt的长度 。
此反应并不需要 AAUAAA序列,
但需要一个识别寡聚 A并指导
poly（ A） 聚合酶延伸的刺激因子 。
The function of polyA
The poly(A) tail is thought to help stabilize
the molecule since a protein binds to it to
resist 3’ exonuclease action and may help in
the translation in the cytoplasm.
This feature has allowed the purification of
mRNA,for cDNA library construction
内含子的剪接 splicing
核酶 (Ribozyme) 1981年 T.Cech和 S.Atman
等 在研究四膜虫 rRNA时发现的
– 一般的酶是纯的蛋白质，而核酶是 RNA或带有蛋白的 RNA；
– 核酶既是催化剂又是底物 。 而酶仅催化反应
核酶发现的意义
– 突破了酶的概念,是一种自体催化；
– 揭示了内含子自我剪接的奥秘；促进了 RNA
的研究。
– 为生命的起源和分子进化提供了新的依据。
Chambon等发现内含子切割位点有 2个特点
（ 1）内含子的两个末端并不存在同源或互补。
这就排除了存在二级结构的可能。
（ 2）连接点具有很短的保守序列，称为 边界顺序 。其规律称为 GT-AG法则 （ GT-AG
rule) 或 Chambon法则 。
左边的剪接位点称 供体 （ donor） 位点,
右边的剪接位点称 受体 （ acceptor） 位点 。
边界顺序
I类内含子的剪接
Cech等 1981年用四膜虫分离得到了 35S的前体
rRNA,它含有一个长 413bp的内含子 。
此 35S rRNA要加入一价或二价阳离子及 GTP
就可以在体外释放出 413b的线性的内含子,
若继续保温,那么线形内含子又可形成环状的
RNA。 这就意味着 35S RNA在 GTP的作用下可以自我剪接 。
I类内含子的结构特点是
1,其边界序列为 5′U-G 3′；
2,具有 中部核心结构 （ Central core
strucature）
3.内部引导顺序 （ internal guide seguence
IGS）
G - O H 内含子 ( 4 1 3 n t ) 水解
P-
外显子 A 外显子 B O H -
5 ’ P - P
L - 1 5 I V S
G - P -P +
+ 4 n t 环状 IVS
- O H
3 9 5 n t
水解
- P
+
G - P - O H L - 1 9 I V S
G - P - O H +
1 5 n t P -
3 9 9 n t
图 1 3 - 四膜虫 3 5 S RN A 内含子剪接 的转酯反应模型
I类内含子的剪切机制
转酯反应 (transesterification)酯键从一个位置转移到另一个位置 。
Ⅱ 类内含子的剪接
(一 )结构特点
(1) 边界序列为
5′↓GUGCG……YnAG↓ ；
(2) 有 6个茎环结构；
有分支点顺序 branch-point
seguence
Ⅱ 类内含子的剪接 机制
无需 鸟苷 的辅助,但需镁离子的存在 。
分枝点 A的 2′-OH对 5′端交界处的磷酸二酯键发动亲核进攻,产生了 套索
（ lariat） 结构 （ 图 13-31） ；
切下的外显子 1其 3′-OH继续对内含子 3′
端的交界序列进行亲核进攻,同时释放出套索状的内含子 。
核 mRNA的剪接
结构特点：
– 边界顺序,符合 GU-AG法则
– 分枝点顺序：为 Py80NPy87Pu75APy95其中 A为百分之百的保守,且具有 2′-OH
– 内含子 5′端有一保守序列可以和 U1 snRNA的
5′端的保守顺序互补
作用特点：需要一些剪接因子 snRNP的参与,U1-U6
剪接因子
snRNPs
U1,U2,U
5和
U4/U6。
P 314
O H
G A G
外显子 1 外显子 2
O H
外显子 1 U G
O
A G
外显子 1 外显子 2
图 13 - 32 核 R N A 的剪接反应 ( 仿 B,L e w i n,
,G E N E S,Ⅵ,199 7,F i g 30.5 )
外显子 内含子
G U U A C A A A C P y AG
5 ’保守顺序 分支位点 P y 区 3 ’保守顺序
U 1 G U U A C A A A C P y AG
U 2 A F
U 2 G U U A C A A A C P y AG
U 5 U 4
U 5 UG
U 6
U 6 U 4 U A C A A A C P y AG
U
G
U A C A A A C P y AG
U
G
U A C A A A C P y AG
U
G
U A C A A A C
G U U A C A A A C P y A G
图 13 - 核 m R N A 剪接反应
E 复合体
U1 结合于 5 ’剪接位点
U 2 A F 结合于 Py 区
A 复合体
U2 结合于分支点
B1 复合体
U 5 / U 4 / U 6 三聚体结合
U5 结合 5 ’端外显子
U6 和 U2 结合
B2 复合体释放 U 1,U 5 从外显子移动到内含子
U6 结合到 5 ’剪接点
C1 复合体释放 U 4,U 6 / U 2 催化转 酯反应
U5 结合于外显子 3 ’剪接位点
5 ’位点被剪切并形成套环
C2 复合体
U 2 / U 5 / U 6 仍结合在套环上释放被剪接的 R N A
套环经去分支被解开
Alternative mRNA processing
可变 mRNA加工
The alternative mRNA processing is the
conversion of pre-mRNA species into more
than one type of mature mRNA
– Alternative selection of poly(A) sites
– Alternative selection of promoters
– Alternative selection of intron or exon
The transcript is polyadenylated at different locations
1 2
3
4 5 6
AATAA AATAA
1 2
3
4
4 5 6321
Calcitonin (thyoid cell 甲状腺 )
CGRP ( brain )
Alternative selection of poly(A) sites
Per-mRNA
polyA2 polyA1
Membrane-anchor
protein in virgin cell
Secreted form in plasma
Immunoglobulin heavy chain:
P1 P2?-amylase in salivary gland
(Stronger splicing)
-amylase in liver
(weaker splicing)
Alternative selection of promoters
P element transposable sysytem in Drosophila
P element
In somatic cell
In germline cell
repressor
transposase
Alternative selection of intron or exon
RNA editing
The sequence of the primary transcript is altered
by either changing,inserting or deleting residues
at the specific points along the molecules is called
RNA editing
The biological significance of editing:
– proofreading校正作用；
– translation regulation调控翻译；
– expanded genetic information扩充遗传信息
They seem to be more common in nonvertebrates.
In human,the unedited mRNA of apolipoprotein
(载脂蛋白 ) B makes correct size of protein in the
liver.
editing
基因组中编码 I S S L C I K V E N L V G V M
D N A 序列 A T A T C A A G T T T A G C T T A T A A A G T A G A G A A C C T G G T A G G T G T A A T
4 8 0 4 0 0 5 0 0 移框 － 1 个碱基
R N A 序列 A U A U C A A G U U U A G G U A U A A A A G U A G A U U G U A U A C C U G G U A G G U G U A A U
蛋白质顺序 I S S L G I K V D C I P G R C N
图 1 3 -4 3 锥虫 co x Ⅱ 基因片断及其产物顺序的比较。核酸序列的数字是以起始密码子 A U G 的 A 开始编号。
蛋白质以单个字母表示。方框表示与酵母、人类同源的氨基酸。黑体表示编辑位点。
( 参考 B,L ew i n,,G E N E S,Ⅵ,1 9 9 7,Fi g 3 1,1 5 )
coxII gene editing in T.brucei
apolipop
rotein
(载脂蛋白 ) B
Mechanics of editing
In1990,the guide RNA was found by L,Simpsom et al.,
Three Mechanisms for mRNA Localization
mRNA Localization
The 3’-UTR Plays an Essential Role
in mRNA Localization
Two Mechanisms of mRNA Degradation
NMD,An mRNA Surveillance System
nonsense-mediated decay (NMD)
Post-Transcriptional Gene Silencing:
RNA Interference (RNAi)
The Mechanism of RNA
Interference (RNAi)
Long double-stranded RNAs (dsRNAs;
typically >200 nt) can be used to silence
the expression of target genes in a variety
of organisms and cell types (e.g.,worms,
fruit flies,and plants),
In mammalian cells,introduction of long
dsRNA (>30 nt) initiates a potent antiviral
response,exemplified by nonspecific
inhibition of protein synthesis and RNA
degradation,The mammalian antiviral
response can be bypassed,however,by the
introduction or expression of siRNAs.
the antisense strand of the siRNA
guides cleavage
The End
Procession
转录后加工
Classes of RNA
Pre-RNA,前体 RNA,The primary transcript has the same
organization as the gene
hnRNA,heterogeneous nuclear RNA,核内不均一 RNA,Eucaryotic
RNA Pol II transcribes a variety of different genes
snRNA,small nuclear RNA,核内小 RNA,is any one of many small
RNA species confined to the nucleus; several of the snRNAs are
involved in splicing or other RNA processing reactions
scRNA,small cytoplasmic RNA,胞内小 RNA,several molecules
present in the cytoplasm and (sometimes) nucleus
rRNA,ribosomal RNA,核糖体 RNA,Serve as components of
ribosomes
tRNA,transfer RNA,转移 RNA,carrying an amino acid; the
covalent linkage is between the NH2 group of the amino acid and
either the 3′- or 2′-OH group of the terminal base of the tRNA t
mRNA,messager RNA,信使 RNA,carries genetic message from
the chromosome to the ribosomes
Function of RNA
Class Function Position
hnRNA 成熟 mRNA的前体 核内
snRNA 参与 hnRNA的剪接、转运 核内
scRNA 蛋白质内质网定位合成的信 号识别体的组成成分 胞内
rRNA 核蛋白体组成成分
tRNA 转运氨基酸
mRNA 蛋白质合成模板
Types of RNA processing
A) Cutting and trimming to generate ends:
– rRNA,tRNA and mRNA
B) Covalent modification:
– Add a cap and a polyA tail to mRNA
– Add a methyl group to 2’-OH of ribose in mRNA and
rRNA
– Extensive changes of bases in tRNA
C) Splicing
– pre-rRNA,pre-mRNA,pre-tRNA by different
mechanisms.
The Long and Winding
Road…..
Eukaryotic and Prokaryotic mRNAs
Have Major Differences - I
Eukaryotic and Prokaryotic mRNAs
Have Major Differences - II
Ribosome核糖体
A supramolecular complex
of rRNAs and proteins,the
site of protein synthesis
Have two subunit
In eukaryotes,binding in
RER; in prokaryotes,
binding in mRNA
一,rRNA processing in
prokaryotes
Seven different operons for rRNA rrn dispersed
thought out the genome
Each operons transcript a 30S(~6000bp) primary
transcript
Each primary transcript contains one copy of
5S,16S and 23S rRNA,together with some tRNA
sequence
Rnase III is involved in rRNA processing
Rnase in E.coli
E.coli中含有的小分子核酸酶酶 基因 底物 功能
RNaseP rnpA tRNA 5′ 端 内切
RnaseO rnpB
RnaseBN? tRNA 3′ 端 外切
RnaseD rnd tRNA 3′ 端 外切
RnaseT? tRNA 3′CCA 外切
RnaseⅢ rnc rRNA和 mRNA 内切
RnaseR? rRNA和 mRNA 外切
RnaseE rne 5S rRNA 内切
RnaseⅠ rna 大部分 RNA 内切
RnaseⅡ rnb RNA 外切多核苷酸磷酸化酶 pnp RNA 外切
RnaseH rnh RNA-DNA杂合链 内切
The primary transcript
Ribonucleoprotein 核糖核酸蛋白复合物 RNP complex
The precursor rRNA folds up
into stem-loop structures by
base pairing in the transcript
Some specific proteins bind to
the rRNA to form RNP complex
Steps in rRNA processing
rRNA前体被大肠杆菌 RNaseⅢ,RNaseE等剪切成一定链长的 rRNA分子
rRNA在修饰酶催化下进行碱基修饰
rRNA与蛋白质结合形成核糖体的大、小亚基
Pre-16S rRNA Pre-5S rRNAPre-t RNA Pre-23S rRNA Pre-tRNA
Precursors
Mature rRNA
Primary
transcript
RNase
Primary rRNA processing in prokaryotes
Eukaryotic rRNA gene organization
In the nucleolus,RNA polymerase I transcribes the
rRNA,which usually exist in tandem repeats
containing 100 or more clusters of the transcription
units
A nucleolus is formed around each cluster
RNA polymerase III transcribes 5S rRNA genes,not in
cluster with other rRNA genes its gene copy number
do not coordinate with other RNA genes;
Primary transcript 47S,containing
– ETS external transcribed sequence外部转录间隔区
– ITS internal transcribed sequence内部转录间隔区
– NTS,non-transcribed spacer非转录间隔区
Some rRNA genes have intron
Eukaryotic rRNA gene organization
rRNA is synthesized in the nucleolus and
assembled with proteins to form subunit
the subunit is then transported into cytoplasm
rRNA processing in eukaryotes
The precursor folds and complexes with proteins
RNP as it is being transcribed in the nucleolus
Many specific ribose methylations take place at
over 100 sites and is carried out directly by a
subset of snRNP particles
snRNPs contain snRNAs that have short streches
of complementarity to parts of the rRNA and,by
base pairing with it,they define where methylation
takes place
pre-rRNA processing is accompanied by the
formation of 60S and 40S subunits
Processing of pre-rRNA
Cleavage is by
ribonucleases,核糖核酸酶,RNA酶
5.8S rRNA is H-
bonded to 28S rRNA
in mature ribosome
Introns in pre-rRNA gene
Only a few organisms’ rRNA gene has introns
Splicing剪接 does not need protein,Ribozyme is
a catalytic RNA molecules that catalyze particular
biochemical reactions in the absence of protein
Introns that does not need proteins for splicing are
categorized as the group I intron
In mRNA,tRNA & rRNA genes in mitochondria
and chloroplasts of yeast/fungi,In rRNA genes of
all insects,In rRNA genes,In some mRNA and
tRNA genes of bacteriophage
Self-splicing自我剪接 for the group I
intron in trtrahymena pre-rRNA
Cleavage and ligation are
two steps here
Cleavage at the 5’ splice
junction,add G at the end
Cleavage at the 3’splice
juntion
Ligation of the exons
Cleavage of the lariat intron
Lariat is an intermediate in
RNA splicing in which a
circular structure with a tail
is created by a 5′-2′ bond
Self-splicing of
trtrahymena pre-rRNA
Need guanosine or
GNP’s 3’-OH
Three phosphoester
bond transfers
Concentration
independent
Not a real enzyme
二,3-D structure of tRNA
Post-transcriptional processing of pre-
tRNA
Addition of the 5’-CCA-3’ to the 3’ end
Chemical modification of the selected
bases,the type and extent of modification
varies
– Specific modification?3D-structure?
–?function (aa-specificity)
Removal of the extra sequences
– 5’-leader sequence,3’-trailer sequence
pre-tRNA
The 5’ and 3’ trailer
sequences are
removed by specific
enzyme during
processing
Processing of eukary,
Pre-tRNA is inside the
nucleus
Modified bases in tRNA
通过甲基化酶，硫醇酶，假尿嘧啶核苷化酶等进行修饰，
如氨基酸臂的 4-硫尿苷（ 4tu）,D臂的 2
甲基鸟苷（ 2mG），
TψC臂的假尿苷（ ψ）
和反密码子环上的 2
异戊腺苷（ 2ipA)
Prokaryote tRNA-genes
A cluster of tRNA genes may be transcribed
into one nascent transcript
5’-leader-(tRNA-spacer)n-tRNA-trailer-3’
Two known enzymes are involved in
processing,RNase P (5’-leader)
and RNase Q (3’-trailer)
Extra enzymes are needed for spacer removal
tRNA processing in prokaryotes
In prokaryotes,mature tRNA are generated
by processing longer pre-tRNA transcripts
by RNases D,E,F and P,followed by
base modification which are unique to each
particular tRNA type
Processing of pre-tRNA
Eukaryote tRNA-genes
真核 tRNA的基因和原核不同
– 真核的前体分子 tRNA是单顺反子，但成 簇排列，基因间有间隔区
– 真核 tRNA基因一般都比原核 tRNA基因多得多，如酵母约有 400个 tRNA基因
– 5′端单磷酸核苷酸，表明已被加工过
– tRNA的前体分子中含有内含子。
Processing of pre-tRNA
tRNA的加工分成 3个阶段
,斩头”，形成 5′末端
去尾，形成 3′-OH末端。缺 -CCA的 tRNA
要用 tRNA核酸转移酶 加 -CCA
修饰：通过甲基化酶，硫醇酶，假尿嘧啶核苷化酶等进行修饰，
4-thiouridine(S4U)4-硫尿苷； inosine(I)次黄嘌呤核苷，肌苷； 1-
methylguanosine(m1G)1-甲基鸟苷； N6-isopentenyladenosine (i6A) N6 -
异戊烯基腺苷； Ribothymidine(T)胸腺嘧啶核糖核苷；
Pseudouridine(ψ)假尿嘧啶核苷； dihydrouridine(D)二氢尿苷真核 tRNA内含子的特点：
位置相同，都在反密码子环的下游
不同 tRNA的内含子长度和序列各异
外显子和内含子交界处无保守序列
内含子的剪切是依靠 RNase异体催化
内含子和反密码子配对形成茎环
RNaseP 核糖核酸酶 P
Ribozyme核酶
RNaseP is endonuclease composed of one RNA
molecules and one protein molecules,It is a simple
RNP
RNaseP are found both in prokaryotes and eukaryotes
The RNA component alone works as an endonuclease,
This RNA is a catalytic RNA,or ribozyme
Ribozyme is a catalytic RNA molecules that catalyze
particular biochemical reactions in the absence of
protein
三,The processing of mRNA
hnRNA Eucaryotic RNA Pol II transcribes a variety of
different genes,from snRNA genes of 60-300nt to the large
Antennapedia genes
The hnRNA is rapidly covered in proteins to form hnRNP
The proteins involved have been classified as hnRNA proteins
A-U
These proteins are organized as a tetramer,Three copies of
tetramer are combined with a RNA molecule of 600-700nt,
called hnRNP particles,each particle has a size of 30-40S
The hnRNP protein are thought to help keep the hnRNA in a
single-stranded form and assist in the various RNA processing
reactions
These hnRNA are processed to give mRNAs called pre-mRNAs
snRNP Particles
snRNAs transcribed by RNA Pol II complex with specific
protein to form snRNP
These RNA are rich in uracil and are denoted as U1,U2 etc
U1,U2,U4,U5 and U6 are those involved in pre-mRNA
splicing,Many others seem to be involved in determining the
sites of methylation of pre-rRNA and are located in the
nulcleolus
The snRNA are synthesized in nucleus by RNA Pol II and
have a normal 5’-cap
They are exported to the cytoplasm where they associate with
the common core protein and other specific protein
Their 5’-cap gains two methyl groups and they are then
imported back into nucleus where they function in splicing
The procession of pre-mRNA
真核 mRNA的加工一般要经过四步：
5′加帽 (capping)
3′加尾 (tailing)
切除内含子 (intron cleavage )
修饰 (modification)：对某些碱基进行 甲基化
5’-capping
The 5’ end is modified by addition of m7G
residue,called a cap,The is nucleotide is added to
the new transcript in the reversed orientation,
giving 5’-5’ linkage
The sugars on the first and second transcribed
nucleotides are methylated,m7GNmpNmp
帽子结构的功能
(1)有助于 mRNA越过核膜，进入胞质；
(2)保护 5′不被酶降解；
(3)翻译时供 IFⅢ （ 起始因子 ） 和核糖体识别 。
加帽
(1) 剪接前加帽如呼肠病毒,牛豆病毒
(2) 剪切后加帽如疱疹病毒和口炎病毒剪接前加帽剪切后加帽帽子 的类型
在末端鸟苷的第 7位上存在单个甲基化位点的称 O型帽子 （ capO） ；
在次末端核苷酸的核糖上的 2′-0位点上还有一个甲基位点的称 1型帽子 (cap 1)；
此外,在第三个核苷酸的核糖上 （ 2′-0） 有甲基化位点的称 2型帽子 （ cap2）
这三种帽子都有特殊 面对面核苷酸结构
（ confrontde nucleotide structure）
三种帽子的共同 在帽 1 中可被甲基化 NH
2
位 置
C N
O C H
3
N C
C H
C N H C C
H N C N N
C H
O C O O O
2
HN
N
N
C H
2
O P O P O P O C H
O O O
帽 1 位 置 O CH
3
O
O P O C H
2
O
帽 2 位 置 O CH
3
O
O P O
O
图 1 3 - m R N A 5 ’ 端的帽子位置和可被甲基化的位点加尾 3’-tailing
成熟的 mRNA是前体 3‘端经过剪切再加上一串多聚 A即
poly(A)尾而形成的。
剪切和聚腺苷酸化需要 DNA和
pro-mRNA上的特定聚腺苷酸化序列 AAUAAA，在其后 11-
20nt处紧随一个 YA结构，在其下游常有一富含 GU的序列。
(1)特殊组分 (CPSF)识别
AAUAAA并指导其它的活性
(2) 剪切因子 (CF)在加尾位点
AAUAAA下游 11－ 30nt 处剪切
RNA；
(3)末端腺苷转移酶 (poly(A)聚合酶 PAP)合成 poly(A)尾巴；
(4) poly(A)结合 蛋白 (PBP)与
poly(A)结合，反应停止。
Polyadenylation signal
聚腺苷酸化信号
5’ Cap…..…..AAUAA..(20bases).CA………UUGUGUUG
signal Poly(A) GUrich region
cleavage site addition site
加 Poly(A)的反应第一步加一个短的寡聚 A序列
（ 10nt ）,此 反 应 依 赖 于
AAUAAA序列；
反应由 poly（ A） 聚合酶在特殊因子指导下完成的 。
第二步是 寡聚 A 尾巴延 伸到
240nt的长度 。
此反应并不需要 AAUAAA序列,
但需要一个识别寡聚 A并指导
poly（ A） 聚合酶延伸的刺激因子 。
The function of polyA
The poly(A) tail is thought to help stabilize
the molecule since a protein binds to it to
resist 3’ exonuclease action and may help in
the translation in the cytoplasm.
This feature has allowed the purification of
mRNA,for cDNA library construction
内含子的剪接 splicing
核酶 (Ribozyme) 1981年 T.Cech和 S.Atman
等 在研究四膜虫 rRNA时发现的
– 一般的酶是纯的蛋白质，而核酶是 RNA或带有蛋白的 RNA；
– 核酶既是催化剂又是底物 。 而酶仅催化反应
核酶发现的意义
– 突破了酶的概念,是一种自体催化；
– 揭示了内含子自我剪接的奥秘；促进了 RNA
的研究。
– 为生命的起源和分子进化提供了新的依据。
Chambon等发现内含子切割位点有 2个特点
（ 1）内含子的两个末端并不存在同源或互补。
这就排除了存在二级结构的可能。
（ 2）连接点具有很短的保守序列，称为 边界顺序 。其规律称为 GT-AG法则 （ GT-AG
rule) 或 Chambon法则 。
左边的剪接位点称 供体 （ donor） 位点,
右边的剪接位点称 受体 （ acceptor） 位点 。
边界顺序
I类内含子的剪接
Cech等 1981年用四膜虫分离得到了 35S的前体
rRNA,它含有一个长 413bp的内含子 。
此 35S rRNA要加入一价或二价阳离子及 GTP
就可以在体外释放出 413b的线性的内含子,
若继续保温,那么线形内含子又可形成环状的
RNA。 这就意味着 35S RNA在 GTP的作用下可以自我剪接 。
I类内含子的结构特点是
1,其边界序列为 5′U-G 3′；
2,具有 中部核心结构 （ Central core
strucature）
3.内部引导顺序 （ internal guide seguence
IGS）
G - O H 内含子 ( 4 1 3 n t ) 水解
P-
外显子 A 外显子 B O H -
5 ’ P - P
L - 1 5 I V S
G - P -P +
+ 4 n t 环状 IVS
- O H
3 9 5 n t
水解
- P
+
G - P - O H L - 1 9 I V S
G - P - O H +
1 5 n t P -
3 9 9 n t
图 1 3 - 四膜虫 3 5 S RN A 内含子剪接 的转酯反应模型
I类内含子的剪切机制
转酯反应 (transesterification)酯键从一个位置转移到另一个位置 。
Ⅱ 类内含子的剪接
(一 )结构特点
(1) 边界序列为
5′↓GUGCG……YnAG↓ ；
(2) 有 6个茎环结构；
有分支点顺序 branch-point
seguence
Ⅱ 类内含子的剪接 机制
无需 鸟苷 的辅助,但需镁离子的存在 。
分枝点 A的 2′-OH对 5′端交界处的磷酸二酯键发动亲核进攻,产生了 套索
（ lariat） 结构 （ 图 13-31） ；
切下的外显子 1其 3′-OH继续对内含子 3′
端的交界序列进行亲核进攻,同时释放出套索状的内含子 。
核 mRNA的剪接
结构特点：
– 边界顺序,符合 GU-AG法则
– 分枝点顺序：为 Py80NPy87Pu75APy95其中 A为百分之百的保守,且具有 2′-OH
– 内含子 5′端有一保守序列可以和 U1 snRNA的
5′端的保守顺序互补
作用特点：需要一些剪接因子 snRNP的参与,U1-U6
剪接因子
snRNPs
U1,U2,U
5和
U4/U6。
P 314
O H
G A G
外显子 1 外显子 2
O H
外显子 1 U G
O
A G
外显子 1 外显子 2
图 13 - 32 核 R N A 的剪接反应 ( 仿 B,L e w i n,
,G E N E S,Ⅵ,199 7,F i g 30.5 )
外显子 内含子
G U U A C A A A C P y AG
5 ’保守顺序 分支位点 P y 区 3 ’保守顺序
U 1 G U U A C A A A C P y AG
U 2 A F
U 2 G U U A C A A A C P y AG
U 5 U 4
U 5 UG
U 6
U 6 U 4 U A C A A A C P y AG
U
G
U A C A A A C P y AG
U
G
U A C A A A C P y AG
U
G
U A C A A A C
G U U A C A A A C P y A G
图 13 - 核 m R N A 剪接反应
E 复合体
U1 结合于 5 ’剪接位点
U 2 A F 结合于 Py 区
A 复合体
U2 结合于分支点
B1 复合体
U 5 / U 4 / U 6 三聚体结合
U5 结合 5 ’端外显子
U6 和 U2 结合
B2 复合体释放 U 1,U 5 从外显子移动到内含子
U6 结合到 5 ’剪接点
C1 复合体释放 U 4,U 6 / U 2 催化转 酯反应
U5 结合于外显子 3 ’剪接位点
5 ’位点被剪切并形成套环
C2 复合体
U 2 / U 5 / U 6 仍结合在套环上释放被剪接的 R N A
套环经去分支被解开
Alternative mRNA processing
可变 mRNA加工
The alternative mRNA processing is the
conversion of pre-mRNA species into more
than one type of mature mRNA
– Alternative selection of poly(A) sites
– Alternative selection of promoters
– Alternative selection of intron or exon
The transcript is polyadenylated at different locations
1 2
3
4 5 6
AATAA AATAA
1 2
3
4
4 5 6321
Calcitonin (thyoid cell 甲状腺 )
CGRP ( brain )
Alternative selection of poly(A) sites
Per-mRNA
polyA2 polyA1
Membrane-anchor
protein in virgin cell
Secreted form in plasma
Immunoglobulin heavy chain:
P1 P2?-amylase in salivary gland
(Stronger splicing)
-amylase in liver
(weaker splicing)
Alternative selection of promoters
P element transposable sysytem in Drosophila
P element
In somatic cell
In germline cell
repressor
transposase
Alternative selection of intron or exon
RNA editing
The sequence of the primary transcript is altered
by either changing,inserting or deleting residues
at the specific points along the molecules is called
RNA editing
The biological significance of editing:
– proofreading校正作用；
– translation regulation调控翻译；
– expanded genetic information扩充遗传信息
They seem to be more common in nonvertebrates.
In human,the unedited mRNA of apolipoprotein
(载脂蛋白 ) B makes correct size of protein in the
liver.
editing
基因组中编码 I S S L C I K V E N L V G V M
D N A 序列 A T A T C A A G T T T A G C T T A T A A A G T A G A G A A C C T G G T A G G T G T A A T
4 8 0 4 0 0 5 0 0 移框 － 1 个碱基
R N A 序列 A U A U C A A G U U U A G G U A U A A A A G U A G A U U G U A U A C C U G G U A G G U G U A A U
蛋白质顺序 I S S L G I K V D C I P G R C N
图 1 3 -4 3 锥虫 co x Ⅱ 基因片断及其产物顺序的比较。核酸序列的数字是以起始密码子 A U G 的 A 开始编号。
蛋白质以单个字母表示。方框表示与酵母、人类同源的氨基酸。黑体表示编辑位点。
( 参考 B,L ew i n,,G E N E S,Ⅵ,1 9 9 7,Fi g 3 1,1 5 )
coxII gene editing in T.brucei
apolipop
rotein
(载脂蛋白 ) B
Mechanics of editing
In1990,the guide RNA was found by L,Simpsom et al.,
Three Mechanisms for mRNA Localization
mRNA Localization
The 3’-UTR Plays an Essential Role
in mRNA Localization
Two Mechanisms of mRNA Degradation
NMD,An mRNA Surveillance System
nonsense-mediated decay (NMD)
Post-Transcriptional Gene Silencing:
RNA Interference (RNAi)
The Mechanism of RNA
Interference (RNAi)
Long double-stranded RNAs (dsRNAs;
typically >200 nt) can be used to silence
the expression of target genes in a variety
of organisms and cell types (e.g.,worms,
fruit flies,and plants),
In mammalian cells,introduction of long
dsRNA (>30 nt) initiates a potent antiviral
response,exemplified by nonspecific
inhibition of protein synthesis and RNA
degradation,The mammalian antiviral
response can be bypassed,however,by the
introduction or expression of siRNAs.
the antisense strand of the siRNA
guides cleavage
The End
