1. 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.

2. RNA Processing And Nuclear Splicing
Timothy G. Standish, Ph. D.

3. Introduction The Central Dogma of Molecular Biology
Cell
DNA
mRNA
Transcription
Reverse tanscription
Polypeptide
(protein)
Translation
Ribosome

4. Eukaryotic Transcription
DNA
Cytoplasm
Nucleus
Nuclear
pores
RNA
Transcription G
AAAAAA
RNA
Processing
mRNA
Export G
AAAAAA

5. A “Simple” Eukaryotic Gene
Transcription
Start Site
5’ Untranslated Region
3’ Untranslated Region
Introns 3’ 5’
Exon 2
Exon 3
Int. 2
Exon 1
Int. 1
Promoter/
Control Region 3’ 5’
Exon 2
Exon 3
Exon 1
Int. 2
Int. 1
Exons
Terminator
Sequence
RNA Transcript

6. Processing Eukaryotic mRNA
3’ Untranslated Region
5’ Untranslated Region
Protein Coding Region 5’ G
5’ Cap 3’ 5’
Exon 2
Exon 3
Int. 2
Exon 1
Int. 1 3’
AAAAA
3’ Poly A Tail
Exon 2
Exon 3
Exon 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

7. 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:
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
Group II introns - Found in organelle and bacterial genes
Differ from Group I introns in sequence and mechanism
Nuclear introns - Found in eukaryotic nuclear genes
Require proteins and other RNAs for splicing

8. Nuclear 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

9. Nuclear Intron SplicingAG GU 3’ 5’
Ex 2
Ex 3
In 2
Ex 1
In 1 3’ 5’
Ex 2
Ex 3
Ex 1
Mutation in AG to AA AA GU 3’ 5’ AG
Ex 2
Ex 3
In 2
Ex 1
In 1 3’ 5’
Ex 3
Ex 1
Mutation in GU to UU GU 3’ AG
Ex 2
Ex 3
In 2 5’ UU
Ex 1
In 1 3’
Ex 2
Ex 3 AG 5’ UU
Ex 1
In 1

10. 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

11. Common Splicing Mechanism
Intron
Exon 2
Exon 1 AG A GU 3’ 5’
U A C U A A C (Yeast)
18-40 BP
Branch site
Left (donor)
5’ splice site
Right (acceptor)
3’ splice site
Py80NPy80Py87Pu75APy95
(Animal-Subscripts indicate percent frequency)
The branch sequence allows identification of the 3’ splice site

12. Common Splicing Mechanism
Folding A OH O HO P AG A G U 3’ 5’ U OH O P G N
Exon 1

13. Common Splicing Mechanism
Lariat Formation A OH O HO P N OH O P - +
Exon 1 G OH O P U OH O P
Transesterification reaction between 2’hydroxyl group on adenine in the branch site and phosphate connecting intron with exon 1

14. Common Splicing Mechanism
Lariat Formation A O HO P OH G OH O P N OH O HO P U OH O P
Exon 1

15. Common Splicing Mechanism
Lariat Formation l
Yee ha!
Exon 2 A AG G U 3’ 5’
Exon 1
Intron
Lariat
Lariat

16. Common Splicing Mechanism
Lariat Removal
Exon 2 A O HO P G N OH
Intron - +
Exon 1 N OH O HO P
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

17. Common Splicing Mechanism
Lariat Removal
Exon 2 O N HO P OH
Exon 1
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

18. Common Splicing Mechanism
Exon 1
Exon 2 5’ 3’ A AG G U
Intron lariat
Following excision, the lariat is rapidly degraded

19. Common Splicing Mechanism
Exon 1
Exon 2 5’ 3’
Following excision, the lariat is rapidly degraded

20. 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

21. The
End