Five Classes of Introns

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Five Classes of Introns
Archaeal introns (tRNAs and rRNAs)
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Generic Splicing Reaction
5 splice junction
3 splice junction
Intron
Exon 2
Exon 1
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
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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

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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

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Group I Intron Structure

• Little conservation of
• primary structure (e.g. P, Q,
• R, S elements, 3splice-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

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Group I Secondary Structure
Internal Guide Sequence (IGS)
G Binding Site (Active Site)
Conserved G
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Group I Intron Splicing Mechanism
G
Autocatalytic or Self-splicing Sequential
Transesterfications Step I 3OH of an
exogenous guanosine attacks the phosphodiester
bond at the 5 splice site -G covalently linked
to intron -5exon now contains a 3 OH group
Step II 3 OH of 5exon attacks
the phosphodiester bond of 3 splice site -intron
is released -exons are ligated together
3 exon
OH
G
5 exon
intron
Step 1
G
G
Step 2
intron
3 exon
5 exon
Joined exons (mature RNA)
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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.

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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

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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 (RGAg) 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)

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Group II Introns
Catalytic Core (Active Site)
Branch Point Adenosine
5 EXON
3 EXON
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Splicing Mechanism for Group II and Pre-mRNA
Introns
Lariat Intermediate
2 to 5 Linkage
3 to 5 Linkage
Phosphate is conserved
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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 gt45 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

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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)

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(No Transcript)
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Pre-tRNA Splicing
Splice
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Splicing of NuclearPre-tRNAIntrons(in Yeast
)Protein-catalyzed1) endonuclease 2)
ligasewith 5 activities
P
Endonuclease
OH
Ligase
or ATP
Kinase
3 phosphodiesterase
Cyclic Phosphodiesterase
Adenylase
Ligase
2'-Phospho transferase
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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

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Bulge-Helix-Bulge Motif

• Two 3 nt bulges on opposite strands separated by
  4 bp

Buldge
Helix
Buldge
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tRNA Processing in Archaea
BHB Endonuclease
Ligase
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rRNA Processing in Archaea
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Inteins Protein Splicing Too!
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Summary of Intron Splicing Mechanisms
Catalytic Mechanisms nucleophiles, introns,
catalysts Splice-site Selection splice
junctions, recognition