1. Five Classes of Introns
Archaeal introns
(tRNAs and rRNAs)

2. Generic Splicing Reaction
5’ splice junction
3’ splice junction
Intron
Exon 1
Exon 2
Intron
Exon 1
Exon 2
Two Steps (“Scissors than Tape”):
Step 1: Break phosphodiester bonds at the exon-intron boundaries
(splice junctions). 5’ bond broken before 3’ bond
Step 2: Formation of a new phosphodiester bond between 3’ end of upstream exon and 5’ end of downstream exon

3. Transesterification
Splicing of Group I, II, and Pre-mRNA introns results from two sequential transesterification reactions
“Transesterification” occurs when a hydroxyl group makes a nucleophilic attack on a phosphodiester bond to form a new phosphodiester bond while displacing a hydroxyl group
The reaction requires no energy (ATP-independent)
Phosphate is conserved

4. Group I Introns Location:
Nuclear rRNA genes of unicellular organisms (e.g. tetrahymena + other ciliates)
Organellar tRNAs and rRNAs (mitochondria and chloroplasts)
rRNA, mRNA, tRNA in bacteria (but rare)
Viruses (e.g. T4 thymidylate synthase mRNA gene)
Not found in vertebrates (e.g. “us”)
Role as Mobile Genetic Elements:
introns can encode homing endonucleases that allow intron mobility

5. Group I Intron Structure
Little conservation of
primary structure (e.g. P, Q,
R, S elements, 3’splice-site G)
All group I introns
fold into a characteristic
secondary structure
(and likely tertiary structure)
X-ray structure has been
solved for most of the intron
from tetrahymena rRNA
RNA folding is critical
for splicing

6. Group I Secondary Structure
Internal
Guide
Sequence
(IGS)
G Binding Site
(Active Site)
Conserved G

7. Group I Intron Splicing Mechanism
Autocatalytic or “Self-splicing”
Sequential Transesterfications:
Step I: 3’OH of an exogenous
guanosine attacks the phosphodiester
bond at the 5’ splice site
-G covalently linked to intron
-5’exon now contains a 3’ OH group
Step II: 3’ OH of 5’exon attacks the
phosphodiester bond of 3’ splice site
-intron is released
-exons are ligated together OH
3’ exon
5’ exon G
intron
Step 1 G G
Step 2
intron
5’ exon
3’ exon
Joined exons (mature RNA)

8. Group II Introns Location: rRNA, tRNA, mRNA Eukaryotic organelles
-mitochondria (fungi), chloroplasts (plants)
mRNA of some Eubacteria (i.e. prokaryotes)
Splicing:
Autocatalytic or self-splicing in vitro
proteins required in vivo
Role as Mobile Genetic Elements:
Introns often encode reverse transcriptases that allow intron to change genomic position.

9. Structure of Group II Introns
Group II introns exhibit little primary sequence conservation
All fold into a common secondary structure containing
six helical domains (d1-d6) that emanate from a “central wheel”
Domains 5 and 6 contain important catalytic activity

10. Tertiary Interactions Critical for Splicing of Group II Introns
Exon binding sequences (EBS 1 and 2) in domain I to intron binding sequences (IBS 1 and 2) near 5’ end of 5’ exon (helps define 5’ splice-site)
Nucleotides in loop of domain 5 interact with nucleotides in domain I
Nucleotides in “wheel” (RGA=g) interact with 3’ splice site (YA= g’) (helps define 3’ splice-site)
Nucleotides in in domain 1(e’) interact with those near 5’ splice site (e)

11. Group II Introns “Catalytic Core” (Active Site) Branch Point Adenosine
5’ EXON
3’ EXON

12. Splicing Mechanism for Group II and Pre-mRNA Introns
Lariat Intermediate
2’ to 5’ Linkage
3’ to 5’ Linkage
Phosphate is conserved

13. Nuclear Pre-mRNA Introns
Location:
Common in vertebrates, numerous introns/gene
Rare in unicellular eukaryotes like yeast, usually one intron/gene when any
Conserved Sequences:
at splice junctions (GT-AG rule), branch site and polypyrimidine tracts
5’ splice site branch site polypyrimidine tract 3’ splice site
yeast AG/GUAUGU UACUAAC Yn CAG/G
metazoans: AG/GURAGU YNCURAC YYYYn YAG/G
A in branch site adenosine is called the branch point
Spacing between the elements is important
The 5’ splice site is generally >45 nucleotides from the branch point
The 3’ splice site is generally nucleotides away from the metazoan branch point and nucleotides from the yeast branch point

14. Pre-mRNA Splicing Requires ~100 proteins and 5 RNAs
Occurs in a large RNP assembly known as the “Spliceosome”
Catalytic component unknown but may be RNA-catalyzed
Splicing via sequential transesterification reactions (same chemical steps as Group II intron splicing)

16. Pre-tRNA Splicing
Splice

17. Splicing of Nuclear Pre-tRNA Introns (in Yeast ) Protein-catalyzed 1) endonuclease 2) ‘ligase’with 5 activities
Endonuclease OH P
Ligase
or ATP
‘Kinase’
‘Cyclic Phosphodiesterase’
3’ phosphodiesterase
‘Adenylase’
‘Ligase’
‘2'-Phospho transferase’

18. Splicing in Archaea tRNAs and rRNAs Endonuclease: -symmetric homodimer
- recognizes/cuts a bulge-helix-bulge motif formed by pairing of region near two exon-intron junctions
Ligase:
- joins exons and circularizes introns

19. Bulge-Helix-Bulge Motif
Two 3 nt bulges on opposite strands separated by 4 bp
Buldge
Helix
Buldge

20. tRNA Processing in Archaea
BHB
Endonuclease
Ligase

21. rRNA Processing in Archaea

22. “Inteins”: Protein Splicing Too!

23. Summary of Intron Splicing Mechanisms
Catalytic Mechanisms: nucleophiles, introns, catalysts
Splice-site Selection: splice junctions, recognition