Foundations of Biology

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Revelation 184 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.

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Nuclear Splicing

• Timothy G. Standish, Ph. D.

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IntroductionThe Central Dogma of Molecular
Biology
Cell
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Eukaryotic Transcription
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A Simple Eukaryotic Gene
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Processing Eukaryotic mRNA
Protein Coding Region

• 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

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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
• Can self splice without the aid of proteins
• Differ from Group I introns in sequence and
  mechanism
• Nuclear introns - Found in eukaryotic nuclear
  genes
• Require proteins and other RNAs for splicing

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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

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Nuclear Intron Splicing
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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

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Common Splicing Mechanism
Py80NPy80Py87Pu75APy95 (Animal-Subscripts
indicate percent frequency)
The branch sequence allows identification of the
3 splice site
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Folding
Common Splicing Mechanism
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Lariat Formation
Common Splicing Mechanism
Transesterification reaction between 2hydroxyl
group on adenine in the branch site and phosphate
connecting intron with exon 1
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Lariat Formation
Common Splicing Mechanism
Exon 1
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Lariat Formation
Common Splicing Mechanism
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Lariat Removal
Common Splicing Mechanism
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
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Lariat Removal
Common Splicing Mechanism
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
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Common Splicing Mechanism
Exon 2
Exon 1
3
5
Following excision, the lariat is rapidly degraded
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Common Splicing Mechanism
Exon 2
Exon 1
3
5
Following excision, the lariat is rapidly degraded
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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

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