Chapter 14 RNA splicing post transcription post-transcription

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Chapter 14 RNA splicing post transcription post-transcription

Chapter 14 RNA splicing

DNA

Pre-mRNA

5’CAP, 3’tail

splicing

i

post-transcriptiontranscription

turnover

mature mRNA

protein


1977 genes in pieces

Evidence for split genes:

Exp1. size of nuclear RNA ≠ cytoplasmic RNA

nuclear RNA 8000 ~10000

NT

short

long

cytoplasmic RNA

2000 NT


利 用 Pulse-chase exp. 來 證 明 precursor-product 之 關 係

標 定 - 追 蹤

1 st step - pulse: freshest RNA transcribed is labeled

Pulse (8000 ~10000 nt)

2 nd step - chase: labeled RNA became processed

Chase (2000 nt)


結 論 :

nuclear RNA (precursor, 10000 nt)

export to cytoplasm

cytoplasmic RNA (mRNA, 2000 nt)


Exp. 2 RNA-DNA hybridization

R-loop: ssDNA 無 法

RNA 配 對 之 處

p401


(heterogeneous nuclear

RNA; hnRNA)

cytoplasmic RNA;

mRNA; mature mRNA)

p401


Splicing

1. The amount of RNA been discarded d d ranging from

50~90%

2. The number of introns per gene varies (0~60)

3. Introns are excised one by one

4. Splicing occurred in the nucleus membrane and

linked to transport through the nucleopores

5. The higher h the euk, the more complex intron


Splicing 機 制

Splicing signal: consensus sequences are important to

proper splicing (p403)

1. 在 交 界 處

exon/GU-intron-AG/exon

2. 在 intron 內

GU-AG rule

-YNCURAC-

a special ilA


Splicing 之 機 制 : 兩 次 phosphotransfer

1 st phosphotransfer

a branched

intermediates

交 界 之 p 由 後 面 exon 提 供

Lariat

2 nd p404


Outline of splicing mechanism

Step 1: 2’-OH of specific A attack exon1-intron boundary

1 st phosphotransfer

form a special 2’→5’ phosphobond (branch point)

Step 2: 3’-OH of exon1 attack exon2-intron boundary

2 nd phosphotransfer

form joint exons and a lariat intron


Evidence: pulse-chase exp

AAAAAAAAAA

CAP * *

* * *

Radioactive pre-mRNA pulse

in vitro splicing (contain

splicing extract)

Stop reaction at different time chase

Analyze by gel electrophoresis


結 果

time 0

漸 增 → product

漸 減 → precursor

先 增 後 減 → intermediate


電 泳 圖

量 化 圖

(intermediate)

( 產 物 )

( 產 物 )

( 產 物 )

( 產 物 )

(intermediate)

Figure 14.5

Time course of intermediate and liberated intron appearance.

p404


Intron 內 含 有 splicing signal

含 specific A 之 conserved 序 列

14.8

p406


Spliceosome: 負 責 進 行 splicing 之 complex

1. 結 構 :RNA & protein form a proper complex

small nuclear RNA

(snRNAs) ~165 NT

Small nuclear ribonuclear

proteins (snRNPs; Snurps)

共 有 U1, 2, 4, 5, 6 五 種 complex

In yeast: 40S (fig 14.9)

In mammal: 60S


U1 & U5: Form perfect complementary to 5’ or 3’

splice site consensus sequence

U2 : Form complementary base pairing to branch-point

consensus sequence

U2AF: U2-associated factor recognizes 3’-spliced site

p407


Base pairing between U1 and 5’ splice site is required for splicing




Base pairing i between U1 and d5’ splice site is not sufficient

i ☺


14.12


p408


U6 can base-pair with h5’ 5’-splice sites

Fig 14.14 A model for interaction between a yeast 5’-

splice site and U6 snRNA

p409


U2 at branch point

U2 snRNA 序 列 可 與 branchpoint A 附 近 序 列 互 補



A


A


A

U


A

U

14.17

p412


U5 lines up 3’ splice site of one exon and

5’ splice site of the next exon

14.21

p416


小 結 : spliceosome 功 能 (p406-421)

1. Form perfect complementary to 5’ or 3’ splice site

consensus sequence i.e. U1, U5 & U6

2. Form complementary base pairing to branch-point

consensus sequence i.e. U2, U6

3. Maintain the structure of spliceosome i.e. U4


Fig 14.27 The spliceosome cycle

p421


Other factors involved in splicing mechanism:

Non-snRNP proteins help association between

snRNP and pre-mRNA

如 : Commitment complex

splicing factors

Use yeast two-hybrid to find proteins associate with

spliceosome (protein-protein interaction) fig 14.32


Fig 14.37 Model for participation of CTD in exon definition

p429


Special events:

1. Alternative splicing: a pre-mRNA can be spliced in

more than one way (fig 14.38, 39, 41)

2Selfsplicing: 2. Self-splicing: splicing occur without spliceosome

(fig 14.48)

3. Trans-splicing: exons are not from the same gene (fig

16.10)

4. RNA editing: genes contain incomplete sequence

information, must be edited to become complete (fig

16.15)


long intron

14.38

結 論 : 一 個 基 因 經 由 alternative splicing 可 產 生 不 同

之 transcripts

p430


The products of Sxl and tra are SR proteins (splicing factors

rich ihin serine and arginine) ii Fig 14.39 Alternative splicing cascade in Drosophila sex determination

p430


Alternative splicing provides a way to get more than

one protein out of the same gene

1 2 3 4 5

6

Fig 14.41 Alternative splicing patterns, coupled with alternative

promoters and polyadenylation sites

p432


Figure 14.48

Self-splicing of Tetraphymena rRNA precursor.

p438


Chapter 16

leader exon

3’

16.10

p489


RNA Editing

16.14

p490


Fig 16.15 Part of the edited sequence of the COIII mRNA of

T. brucei

p490


DNA

Pre-mRNA

5’CAP, 3’tail

splicing

post-transcription

transcription

turnover

mature mRNA

protein ti


Posttranscriptional control of gene expression (p494~517)

A very important stage of gene regulation in eukaryotes

1. Casein mRNA stability (table 16.1)

2. Transferrin receptor mRNA stability (fig 16.31)

3 RNA interference (RNAi): post transcriptional gene

3. RNA interference (RNAi): post-transcriptional gene

silencing (fig 16.39)


結 論 :prolactin 選 擇 性 的 增 加 casein mRNA stability

4X

1.3X

26X

p495


Factors that are important for the stability of

mRNA:

1. 5’CAP and 3’ polyA tail

2. 3’ untranslated region (3’-UTR) fig 16.27

5’ 3’

coding region

AAAAAAAAAA

CAP

5’UTR

3’UTR


conclusion: deletion of 3’-UTR, lose response to iron

coding

3’-UTR (~2.6 kb)

Fig 16.21 Effects of 3’-UTR on the iron-responsiveness of cell surface concentration of TfR

TfR gene: transferrin receptor

Cell 缺 鐵 TfR expression↑; cell 不 缺 鐵 :TfR expression↓

p496


5 stem loops (A~E) in 3’-UTR of TfR mRNA called iron

response element (IRE)

Fig 16.22, p496


結 論 :3’-UTR deletion or mutation, alter the response to Fe

+Fe -Fe +Fe -Fe +Fe -Fe

loss Fe response, mRNA stable

loss Fe response, mRNA unstable

Fig 16.25 Effects on iron responsiveness of deletions in the IRE region of the TfR 3’-UTR

Fig 16.25; p498


RNA interference (RNAi); post-transcriptional gene

silencing (PTGS): control of gene expression by specific

mRNA degradation caused by dsRNA

Fig 16.28 p501


Negative

control

Positive

control

antisense

RNA

ds RNA

Fig 16.29 ds RNA-induced RNAi causes destruction of a specific mRNA

p501


siRNA

能 進 行 RNAi

結 論 :dsRNA is degraded into short pieces (21-23 nt)

Fig 16.30, p502


*

yield shortest fragment

結 論 :dsRNA determines the

cleavage sites of target mRNA

Fig 16.31, p503


結 論 :cleavage occurs at

21-23nt 23nt interval

Fig 16.32, p503


RNA interference 之 機 制

trigger

(RNase)

short interfering

target

RISC: RNA-induced silencing

complex

Fig 16.33, p538


Amplification of siRNA: increase the sensitivity of RNAi

( 含 RNase III 及 RNA helicase 活 性 )

RNA-directed RNA pol

Fig 16.38, p508


microRNA (miRNA): small

RNAs produced naturally

Fig 16.45 Two pathways to gene silencing by miRNAs

p516


DNA

transcription

i

5’CAP, 3’tail

splicing

Pre-mRNA

post-transcription

transcription

turnover

mature mRNA

protein

合 成 速 率

transcription

Steady state

mRNA pool in

cytoplasm 累

積 量

分 解 速 率

Post-transcription


Increase

transcription

activity

合 成 速 率 變 快

Increase

steady state

mRNA pool

累 積 量 變 大

Slower rate of

mRNA

degradation

分 解 速 率 變 慢

合 成 速 率 變 慢

分 解 速 率 變 快

累 積 量 變 小


How to estimate the effect of transcription and posttranscription

regulation

Nuclear run-on transcription (fig 5.33; p112)

Isolate nuclei→isolate run-

on transcript (hnRNA)

→estimate amount of a

particular hnRNA

Isolate total mRNA (steadyso

e o N (s e dy

state RNA)→estimate

amount of a particular

mRNA


Amount of Amount of

hRNA hnRNA of gene A mRNA of gene A

實 驗 組 100 90

對 照 組 10 9

比 例 10X ↑ 10X ↑

結 論 :transcriptional control determines gene A expression

Amount of Amount of

hnRNA of gene B

mRNA of gene B

實 驗 組 100 10

對 照 組 100 100

比 例 1X 10X ↓

結 論 :post-transcriptional control determines gene B expression


Fig 14. 1, 2, 4, 5, 8, 10, 12, 14, 17, 21, 27,

37~39, 41, 48

Fig 16. 10, 14, 15, 21, 22, 25, 28~33 33, 38,

45

Table 16.1

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