Five Classes of Introns
Archaeal introns
(tRNAs and rRNAs)
Generic Splicing Reaction
5’ splice junction
Exon 1
Intron
3’ splice junction
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
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
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
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
Group I Secondary Structure
Internal
Guide
Sequence G Binding Site
(IGS)
(Active Site)
Conserved G
Group I Intron Splicing Mechanism
GOH
3’ exon Autocatalytic or “Self-splicing”
G
5’ exon
intron
Step 1
G
G
Step 2
intron
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
5’ exon 3’ exon
Joined exons (mature RNA)
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.
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
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)
Group II Introns
“Catalytic
Core”
(Active
Site)
Branch Point
Adenosine
5’
EXON
3’
EXON
Lariat Intermediate
2’ to 5’ Linkage
Splicing
Mechanism for
Group II and
Pre-mRNA
Introns
3’ to 5’ Linkage
Phosphate is conserved
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
yeast
metazoans:
5’ splice site
AG/GUAUGU
AG/GURAGU
branch site polypyrimidine tract
UACUAAC
Yn
YNCURAC
YYYYn
3’ splice site
CAG/G
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 18-38 nucleotides away from the metazoan branch
point and 6-150 nucleotides from the yeast branch point
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)
Pre-tRNA Splicing
Splice
P
OH
Endonuclease
Ligase
‘Kinase’
3’ phosphodiesterase
‘Cyclic Phosphodiesterase’
‘Adenylase’
‘Ligase’
‘2'-Phospho transferase’
or ATP
Splicing of
Nuclear
Pre-tRNA
Introns
(in Yeast )
Protein-catalyzed
1) endonuclease
2) ‘ligase’with 5
activities
Splicing in Archaea
• tRNAs and rRNAs
• Endonuclease:
-symmetric homodimer
- recognizes/cuts a bulge-helix-bulge motif
formed by pairing of region near two exonintron junctions
• Ligase:
- joins exons and circularizes introns
Bulge-Helix-Bulge Motif
• Two 3 nt bulges on
opposite strands
separated by 4 bp
Buldge
Helix
Buldge
tRNA Processing in Archaea
BHB
Endonuclease
Ligase
rRNA Processing in Archaea
“Inteins”: Protein Splicing Too!
Summary of Intron Splicing Mechanisms
Catalytic Mechanisms: nucleophiles, introns, catalysts
Splice-site Selection: splice junctions, recognition