Revelation 18:4
4 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.
©2000 Timothy G. Standish
Nuclear Splicing
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
Introduction
The Central Dogma
of Molecular Biology
Cell
Transcription
Translation
Reverse
tanscription DNA
mRNA
Ribosome
Polypeptide
(protein)
Eukaryotic Transcription
Cytoplasm
DNA
Transcription
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
AAAAAA
Export
©2000 Timothy G. Standish
A “Simple” Eukaryotic Gene
Transcription
Start Site
3’ Untranslated Region
5’ Untranslated Region
Introns
5’
Exon 1 Int. 1
Promoter/
Control Region
Exon 2
3’
Int. 2 Exon 3
Terminator
Sequence
Exons
RNA Transcript
5’
Exon 1 Int. 1
Exon 2
Int. 2 Exon 3
3’
©2000 Timothy G. Standish
Processing Eukaryotic mRNA
5’ Untranslated Region
3’ Untranslated Region
Protein Coding Region
5’5’ G
3’
Int. 11 Exon
Int. 23 Exon 3 AAAAA 3’
Exon 1Exon
Exon22 Exon
5’ Cap
3’ Poly A Tail
RNA processing achieves three things:
 Removal of introns
 Addition of a 5’ cap
 Addition of a 3’ tail
l
This signals the mRNA is ready to move out of
the nucleus and may control its life span in the
cytoplasm
©2000 Timothy G. Standish
Introns
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
©2000 Timothy G. Standish
Nuclear Intron Splicing
Exon/intron junctions have short but well
conserved consensus sequences
The generic sequence of an intron is:
GT . . . AG in DNA or GU . . . AG 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
©2000 Timothy G. Standish
Nuclear Intron Splicing
5’
Ex 1
GU
AG
In 1
5’
Mutation in AG to AA
Ex 1
In 1
5’
GU
Ex 1
Ex 2
Ex 2
AA
Ex 2
5’
Mutation in GU to UU
Ex 1
In 1
5’
UU
AG
Ex 2
5’
AG
Ex 2
Ex 1
UU
In 1
GU
Ex 1
In 2
In 2
AG
Ex 3
3’
AG
Ex 3
3’
3’
Ex 3
GU
3’
3’
Ex 3
GU
Ex 3
AG
In 2
Ex 3
3’
©2000 Timothy G. Standish
Splicing Order
Some gene transcripts have been shown to
loose their introns in a consistent order
The 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
©2000 Timothy G. Standish
Common Splicing Mechanism
Exon 1
5’
Intron
GU
A
Left
(donor)
5’ splice
site
Branch
site
18-40 BP
AG
Exon 2
3’
Right
(acceptor)
3’ splice
site
U A C U A A C (Yeast)
Py80NPy80Py87Pu75APy95
(Animal-Subscripts indicate percent frequency)
The branch sequence allows
identification of the 3’ splice site
©2000 Timothy G. Standish
Common Splicing Mechanism
Folding
O
HO P
O
O
U
O
O P
A
O
A
G
AG
3’
OH
OH
5’
Exon 1
©2000 Timothy G. Standish
Common Splicing Mechanism
O
HO P
O
O
O
O P
O
OH
-
A
Lariat
Formation
-OH+
- +-
Exon 1
Transesterification reaction between
2’hydroxyl group on adenine in the
branch site and phosphate connecting
intron with exon 1
©2000 Timothy G. Standish
Common Splicing Mechanism
O
HO P
O
O
A
Lariat
Formation
O
O P
O
OH
Exon 1
©2000 Timothy G. Standish
Common Splicing Mechanism
Lariat
Formation
Yee ha!
Lariat
l
Intron
U
A
G
AG
Exon 2
3’
l
©2000 Timothy G. Standish
O
O
HO P
OH
O
HO P
+- +
O
Lariat
Removal
O
-
O
OH
G
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
O
O
HO P
O
OH
A
O
HO P
O
O
Intron
N
Common Splicing Mechanism
Exon 2
Exon 1
©2000 Timothy G. Standish
O
O
HO P
OH
Lariat
Removal
O
O
O
HO P
O
O
HO P
O
O
Exon 1
OH
N
N
Common Splicing Mechanism
Exon 2
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
©2000 Timothy G. Standish
Common Splicing Mechanism
Exon 1
Exon 2
5’
3’
Intron lariat
Following
excision, the
lariat is rapidly
degraded
©2000 Timothy G. Standish
Common Splicing Mechanism
Exon 1
5’
Exon 2
3’
Following
excision, the
lariat is rapidly
degraded
©2000 Timothy G. Standish
The 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
©2000 Timothy G. Standish
©2000 Timothy G. Standish