GPLS 701 Advanced Molecular Biology RNA Processing

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GPLS 701 Advanced Molecular Biology RNA
Processing

• Gerald M. Wilson, Ph.D.
• Department of Biochemistry and Molecular Biology
• BRF Room 239
• gwils001_at_umaryland.edu

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Gene Expression in Eukaryotes
(Orphanides and Reinberg (2002) Cell 108, 439-451)
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Identification of introns by R-looping
Nelson Cox, 2000, p. 992
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Net effect of nuclear pre-mRNA splicing
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In vitro splicing reactions

• General requirements
• 32P-labeled pre-mRNA substrate
• source of splicing factors (recombinant and/or
  cellular)
• ATP
• Much of our current knowledge of splicing has
  been derived from addition/depletion studies
  using these systems

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General features of nuclear introns
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General mechanisms for nuclear pre-mRNA splicing
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The lariat is formed by transesterification
between the 5-end of the intron sequence and the
2-OH of the branch site adenosine
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Pre-mRNA splicing is mediated by a large
multicomponent complex(the spliceosome)
(Figure 14.13 second edition)
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Trans-acting factors participating in pre-mRNA
splicing

• Small nuclear ribonuclear proteins (snRNPs, or
  Snurps)
• U1, U2, U4, U5, and U6
• 3-splice site selectors
• ie Slu7, U2AF
• SR proteins
• ie SC35, SF2/ASF
• Splicing regulatory proteins (not to be confused
  with SR proteins, although some are)
• ie Tra2, Sam68

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snRNPs in the spliceosome cycle
Figure 14.28
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Spliceosome assembly
Figure 14.26
U1 is first!
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U1 snRNA defines the 5-splice junction by base
complementarity
Figure 14.12
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Figure 14.13
Figure 14.11
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U2 snRNA defines the branchpoint A by base
complementarity
Figure 14.17
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U4, U5, and U6 snRNPs

• Recruited to the spliceosome as a complex (the
  U4/U6U5 tri-snRNP)
• U4 is released in the active spliceosome (likely
  functions to sequester U6 until needed)
• U6 and U5 are collectively responsible for
  coordinating the 5- splice site, the branch
  point A, and the 3-splice site in the active
  complex
• NB specificity for most interactions between
  snRNPs is mediated by formation of RNA base
  pairs, while proteinprotein contacts contribute
  stability to some complexes

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U4 and U6 snRNPs are linked by base
complementarity
Figure 14.22
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U6 snRNA interactions
5-end of intron (replaces U1 in the active
complex)
Figure 14.14
Together, these interactions juxtapose the splice
donor (5-end of intron) and the branch point.
U2 snRNA
From Figure 14.23
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U5 snRNA interactions
Cross-linking indicates that U5 snRNA is in close
proximity to the 3-end of the upstream exon (E1)
and the 5-end of the downstream exon (E2)
Figure 14.23
These interactions bring the 5- and 3-ends of
the intron together in the active spliceosome
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Selection of the 3-splice site
Slu7, U2AF are required for selection of the
correct splice acceptor site.
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SR proteins and commitment

• Commitment occurs when a splice site has been
  targeted by a sequence-specific RNA-binding
  protein, which in turn induces spliceosome
  assembly.
• SR proteins are RNA-binding proteins containing
  an Arg/Ser-rich domain (RS domain) that often
  serve to commit splicing of specific introns.
• SR proteins may also bind RNA sequences known as
  splicing enhancers, however, additional factors
  which are not SR proteins may also bind these
  sites.

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How can SR proteins contribute to regulation of
splicing?

• bridging 3-site selectors and U1 snRNP
• compensating weak 3-sites by binding exonic
  splicing enhancer sequences
• regulating splice site selection by competing
  with splicing inhibitors

(Hastings and Krainer (2001) Curr. Opin. Cell
Biol. 13, 302-309)
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Exon 5 of CD44 is alternatively spliced during
cellular stress responses
(Matter et al (2002) Nature 420, 691-695)
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Inclusion of CD44 exon 5 is normally repressed by
hnRNP A1 binding to an exonic splicing silencer
splicing activators
splicing inhibitor
exonic splicing silencer
exonic splicing enhancer
(Shin and Manley (2004) Nat. Rev. Mol. Cell Biol.
5, 727-738)
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Ras activation may promote inclusion of CD44 exon
5 by Sam68-dependent occlusion of the hnRNP A1
binding site
(Shin and Manley (2004) Nat. Rev. Mol. Cell Biol.
5, 727-738)
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Group I and II introns

• found in some nuclear, mitochondrial, and
  chloroplast genes coding for rRNA, mRNA, and
  tRNA
• no external source of energy (ie ATP) required
  for intron excision
• self-splicing (ie no protein enzymes required)
• transesterification reaction in Group I introns
  initiated by a guanine nucleotide or nucleoside,
  while in Group II, an internal branch site A is
  used (generating a lariat product)
• discovery of Group I and II introns provided
  one of the first demonstrations of the existence
  of catalytic RNA

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Ribozymes

• RNA molecules capable of catalyzing biochemical
  reactions
• Earliest known examples
• RNase P
• Group I and II introns
• hammerhead ribozymes
• Principal reactions
• RNA transesterification
• RNA cleavage (hydrolysis of phosphodiester
  bonds)
• Substrate aligned into the active site using a
  guide sequence which is complimentary to the
  substrate
• All ribozymes depend absolutely on the assumption
  of correct 3-dimensional structure for activity

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Cleavage site of the hammerhead ribozyme
Nelson Cox, 2005, p. 1018
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Group I self-splicing rRNA intron from Tetrahymena
Nelson Cox, 2005, p. 1018

• Features
• internal guide sequence (yellow box)
• 5 intronexon junction (red arrow)
• 3 intronexon junction (blue arrow)
• sequences near intronexon junctions are
  complementary to guide sequence (green)

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Group I self-splicing rRNA intron from
Tetrahymena contd
Guo et al. (2004) Mol. Cell 16, 351
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The mRNA 5-cap structure

• Functions
• essential for nucleo-cytoplasmic transport of
  mRNAs through interaction with nuclear
  cap-binding proteins
• increases the efficiency of translation by
  targeting formation of the preinitiation complex
  (cytoplasmic cap-binding proteins)
• protects the transcript from 5?3
  exoribonucleolytic activities

Nelson Cox, 2005, p. 1008
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Enzymatic reactions required for mRNA 5-capping
Nelson Cox, 2005, p. 1008
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Sequence determinants of 3 mRNA processing
20-40 nucleotides apart
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3 mRNA processing in eukaryotes
(1) An enzyme complex recognizes the
polyadenylation signal (AAUAAA) and a less well
conserved G-U rich sequence located 20-40
nucleotides downstream. (2) An endonuclease
cleaves the primary transcript 10-30 nucleotides
downstream of the AAUAAA signal. (3) A series
of 80-250 A residues are added to the 3-end of
the cleaved transcript by polyadenylate
polymerase.
Nelson Cox, 2005, p. 1013
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Multi-subunit protein complexes direct mRNA
3-cleavage and polyadenylation
(Proudfoot (2004) Curr. Opin. Cell Biol. 16,
272-278)
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Functions of the mRNA 3 poly(A) tail

• The 3 poly(A) tail is bound by a linear array of
  poly(A)-binding proteins. This interaction
  serves to
• Increase translation efficiency by complexing
  with eIF4G in the pre-initiation complex
• Protect the transcript from 3?5 exonucleases

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Synthesis and processing of ovalbumin mRNA
Nelson Cox, 2005, p. 1013
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Differential RNA processing Multiple mRNAs from
a single gene
Nelson Cox, 2005, p. 1014
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Tissue-specific processing of the calcitonin
primary transcript
Nelson Cox, 2005, p. 1015