Revelation 18:4
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©2001 Timothy G. Standish
Revelation 18:44 And I heard another voice
from heaven, saying, Come out of her, my people, that ye be not partakers of her sins, and that ye receive not of her plagues.
©2001 Timothy G. Standish
RNA ProcessingRNA ProcessingAnd Nuclear SplicingAnd Nuclear Splicing
Timothy G. Standish, Ph. D.
mRNA
Transcription
IntroductionIntroduction
The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology
Cell
Polypeptide(protein)
TranslationRibosome
Reverse tanscription DNA
©2001 Timothy G. Standish
DNA
Cytoplasm
Nucleus
Eukaryotic TranscriptionEukaryotic Transcription
ExportG AAAAAA
RNA
Transcription
Nuclear pores
G AAAAAA
RNAProcessing
mRNA
©2001 Timothy G. Standish
3’5’ Exon 2 Exon 3Exon 1 Int. 2Int. 1
A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene
Terminator Sequence
Promoter/Control Region
Transcription Start Site
RNA Transcript
5’ Untranslated Region 3’ Untranslated Region
Exons
Introns
3’5’ Exon 2 Exon 3Int. 2Exon 1 Int. 1
©2001 Timothy G. Standish
3’5’ 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
Int. 2
Int. 1
RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail
This signals the mRNA is ready to move out of the nucleus and may control its life span in the cytoplasm
©2001 Timothy G. Standish
IntronsIntrons Introns are intervening sequences that “interrupt” eukaryotic genes and must be removed before uninterrupted exons coding for proteins leave the nucleus as mRNA
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
©2001 Timothy G. Standish
Nuclear Intron SplicingNuclear Intron Splicing Exon/intron junctions have short but well
conserved consensus sequences The generic sequence of an intron is: GTNN . . . NNAG in DNA or GUNN . . . NNAG in
RNA This sequence does not apply to the introns of
organelles or yeast tRNA genes 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 intron will be spliced out
The splicing apparatus is usually not tissue specific
©2001 Timothy G. Standish
Mutation in GU to UUGU 3’AGEx 2 Ex 3In 2AG5’ UUEx 1 In 1
Mutation in AG to AA
Nuclear Intron SplicingNuclear Intron Splicing
3’Ex 2 Ex 3AG5’ UUEx 1 In 1
3’5’ Ex 2 Ex 3Ex 1
AG GU 3’5’ AGGU Ex 2 Ex 3In 2Ex 1 In 1
3’5’ Ex 3Ex 1
AA GU 3’5’ AGGU Ex 2 Ex 3In 2Ex 1 In 1
©2001 Timothy G. Standish
Splicing OrderSplicing OrderSome gene transcripts have been shown
to loose their introns in a consistent orderThe current model says that the hnRNA
adopts different conformations after specific introns are removed thus making other introns available for removal
Thus, the removal of introns does not proceed sequentially along the transcript
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing Mechanism
Exon 2Exon 1Intron
AGAGU 3’5’18-40 BP
Branch site
Left (donor)5’ splice
site
Right (acceptor)3’ splice
site
Py80NPy80Py87Pu75APy95 (Animal-Subscripts indicate percent frequency)
U A C U A A C (Yeast)
The branch sequence allows identification of the 3’ splice site
©2001 Timothy G. Standish
AGA GU
3’
5’
Common Splicing MechanismCommon Splicing MechanismFoldingFolding
U
OH
O
O
O
OP G
OH
O
O
O
O
OP
O
O
O
P
N
OH
O
O
O
O
P
A
OHO
HO
O
O
OP
O
OH
O
P
Exon 1
©2001 Timothy G. Standish
Exon 1
Common Splicing MechanismCommon Splicing Mechanism
G
OH
O
O
O
O
OP
O
O
O
P
A
OHO
HO
O
O
OP
O
OH
O
P
N
OH
O
O
O
O
P
U
OH
O
O
O
OP
++
--
-
-
-
Lariat Lariat FormationFormation
Transesterification reaction between 2’hydroxyl group on adenine in the branch site and phosphate connecting intron with exon 1
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing Mechanism
G
OH
O
O
O
O
OP
O
O
O
P
A
O
HO
O
O
OP
O
OH
O
P
U
OH
O
O
O
OP
N
OH
O
O
O
HO
P
O
Lariat Lariat FormationFormation
Exon 1
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing MechanismLariat FormationLariat Formation
Exon 2A AG
GU
3’
5’
3’
Exon 1Intron
Lariat
Yee ha!Lariat
©2001 Timothy G. Standish
++
Common Splicing MechanismCommon Splicing MechanismLariat Lariat RemovalRemoval
Exon 1
N
OH
OO
O
HO
P
O-
-
--
-
A second nucleophilic transesterification reaction, this time between 3’ hydroxyl group on nucleotide 1 in exon 1 and the phosphate connecting intron 2 with exon 2
Exon 2
A
O
HO
OO
OP
G
O
HO
O
OP
N
O
HO
O
OP
HO
O
OP
OH
OH OH
Intron
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing MechanismLariat Lariat RemovalRemoval
A second nucleophilic transesterification reaction, this time between 3’ hydroxyl group on nucleotide 1 in exon 1 and the phosphate connecting intron 2 with exon 2
Exon 2
O
N
O
HO
O
OP
N
O
HO
O
OP
HO
O
OP
OH
OH
Exon 1
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing Mechanism
3’
Exon 2Exon 15’
A
AG
GU
Intron lariat
Following excision, the lariat is rapidly degraded
©2001 Timothy G. Standish
Common Splicing MechanismCommon Splicing Mechanism
3’
Exon 2Exon 15’
Following excision, the lariat is rapidly degraded
©2001 Timothy G. Standish
The SpliceosomeThe Spliceosome Spliceosomes are structures that form within
the nucleus to remove introns from eukaryotic hnRNA
This structure is large, on the order of a ribosome subunit
Like the ribosome, spliceosomes are composed of both protein and RNA
©2001 Timothy G. Standish
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