Nuclear Splicing by devender

8/14/2019 Nuclear Splicing by devender

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2000 Timothy G. Standish

Nuclear SplicingNuclear Splicing

Prepared by : Devender Arora

STUDENT : UIET MDU ROHTAK


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mRNA

Transcription

IntroductionIntroduction

The Central DogmaThe Central Dogmaof Molecular Biologyof Molecular Biology

Cell

Polypeptide(protein)

Translation Ribosome

Reverse tanscription DNA


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DNA

Cytoplasm

Nucleus

Eukaryotic TranscriptionEukaryotic Transcription

ExportG AAAAAA

RNA

Transcription

Nuclearpores

G AAAAAA

RNAProcessing

mRNA


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A Simple Eukaryotic GeneA Simple Eukaryotic Gene

TerminatorSequence

Promoter/ Control Region

TranscriptionStart Site

RNA Transcript

5 Untranslated Region 3 Untranslated Region

Exons

Introns

35 Exon 2 Exon 3Int. 2Exon 1 Int. 1

35 Exon 2 Exon 3Exon 1 Int. 2Int. 1


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35 Exon 2 Exon 3Int. 2Exon 1 Int. 1

Processing Eukaryotic mRNAProcessing Eukaryotic mRNA

Protein Coding Region

3 Untranslated Region5 Untranslated Region

3

AAAAA

3 Poly A Tail

5 G

5 Cap

Exon 2 Exon 3Exon 1

I n t

. 2

I n t

. 1

q RNA processing achieves three things:1 Removal of introns2 Addition of a 5 cap3 Addition of a 3 tail

q This signals the mRNA is ready to move out of the nucleus and may control its life span in thecytoplasm


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IntronsIntronsq Introns are intervening sequences that interrupt eukaryotic genes and must be

removed before uninterrupted exons coding for proteins leave the nucleus as mRNA

q Three types of intron are known:1 Group I introns - Found in organelle and bacterial genes along with some lower

eukaryotes nuclear genes- Can self splice without the aid of proteins

- Require free GTP for splicing

2 Group II introns - Found in organelle and bacterial genes- Can self splice without the aid of proteins

- Differ from Group I introns in sequence and mechanism

3 Nuclear introns - Found in eukaryotic nuclear genes- Require proteins and other RNAs for splicing


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Nuclear Intron SplicingNuclear Intron Splicingq Exon/intron junctions have short but well conserved consensus

sequencesq The generic sequence of an intron is:q GT . . . AG in DNA or GU . . . AG in RNAq This sequence does not apply to the introns of organelles or yeast

tRNA genesq Splice sites operate in pairs which are generic. Thus, if the end of one

intron is mutated, that intron plus the following exon and next intronwill be spliced out

q The splicing apparatus is usually not tissue specific



Mutation in GU to UUGU 3AGEx 2 Ex 3In 2AG5 UUEx 1 In 1

Mutation in AG to AA

Nuclear Intron SplicingNuclear Intron Splicing

3Ex 2 Ex 3AG5 UUEx 1 In 1

35 Ex 2 Ex 3Ex 1

AG GU 35 AGGU Ex 2 Ex 3In 2Ex 1 In 1

35 Ex 3Ex 1

AA GU 35 AGGU Ex 2 Ex 3In 2Ex 1 In 1



Splicing OrderSplicing Orderq Some gene transcripts have been shown to loose

their introns in a consistent orderq The current model says that the hnRNA adopts

different conformations after specific introns are

removed thus making other introns available forremoval

q Thus, the removal of introns does not proceed

sequentially along the transcript



Common Splicing MechanismCommon Splicing Mechanism

Exon 2Exon 1 IntronAGAGU 35

18-40 BP

Branchsite

Left

(donor)5 splicesite

Right

(acceptor)3 splicesite

Py 80 NPy 80 Py 87 Pu 75 APy 95 (Animal-Subscripts indicate percent frequency)

U A C U A A C (Yeast)

The branch sequence allows

identification of the 3 splice site



AGAG

U

3

5

Common Splicing MechanismCommon Splicing MechanismFoldingFolding

U

O H

O O

O O

P G

O H

O O

O

O O

P

O O

O P

N

O H

O

O O

O P

A

OHO

HO

O

O

OP

OOH

O

P

Exon 1



Exon 1


G

O H

O O

O

O O

P

O O

O P

A

OHO

HO

O

O

OP

OOH

O

P

N

O H

O

O O

O P

U

O H

O O

O O

P

++-

--

-

-

LariatLariat

FormationFormation

Transesterification reaction between2hydroxyl group on adenine in the

branch site and phosphate connectingintron with exon 1




G

O H

O O

O O

O

P

O O

O P

A

O

HO

O

O

OP

OOH

O

P

U O H

O O

O O

P

N

O H

O

O O

H O

P O

LariatLariat

FormationFormation

Exon 1



Common Splicing MechanismCommon Splicing MechanismLariatLariat

FormationFormationExon 2A AGG

U

3

5

3

E x o n 1

Intron

Lariat

Yee ha!Lariat



++


RemovalRemoval

Exon 1

N

O H

O O

O

H O

P

O

--

--

-

A second nucleophilictransesterification reaction, thistime between 3 hydroxyl groupon nucleotide 1 in exon 1 andthe phosphate connecting intron2 with exon 2

Exon 2

A

O

H O

O O

O

P

G

O

H O

O

O

P

N

O

H O

O

O

P

H O

O

O

P

O H

O H O

H

Intron




RemovalRemoval

A second nucleophilictransesterification reaction, thistime between 3 hydroxyl groupon nucleotide 1 in exon 1 andthe phosphate connecting intron2 with exon 2

Exon 2 O

N

O

H O

O

O

P

N

O

H O

O

O

P

H O

O

O

P

O H

O H

Exon 1




3Exon 2Exon 1

5

A

A G

G

U

Intron lariat

Followingexcision, thelariat is rapidlydegraded




3Exon 2Exon 1

5

Followingexcision, thelariat is rapidlydegraded



The SpliceosomeThe Spliceosomeq Spliceosomes are structures that form within the

nucleus to remove introns from eukaryotic hnRNAq This structure is large, on the order of a ribosome

subunitq Like the ribosome, spliceosomes are composed of

both protein and RNA


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Nuclear Splicing by devender

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