RNA Processing and Turnover Gerald Wilson, Ph'D' gwils001umaryland'edu

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RNA Processing and TurnoverGerald Wilson,
Ph.D.gwils001_at_umaryland.edu
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Major RNA processing mechanisms in eukaryotes

• ribosomal RNA (RNA polymerase I transcripts)
• (i) methylation
• (ii) nucleolytic cleavage
• messenger RNA (RNA polymerase II transcripts)
• (i) 5-capping
• (ii) 3-cleavage and polyadenylation
• (iii) removal of introns (nuclear splicing)
• transfer RNA (RNA polymerase III transcripts)
• (i) 5- and 3-cleavage
• (ii) CCA addition
• (iii) base modification
• (iv) tRNA splicing

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Major RNA processing mechanisms in prokaryotes

• ribosomal RNA
• (i) methylation
• (ii) nucleolytic cleavage
• messenger RNA
• no significant modification
• transfer RNA
• (i) 5- and 3-cleavage
• (ii) CCA addition
• (iii) base modification

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Messenger RNA processing in eukaryotes

• Nuclear splicing
• 5-capping
• 3-cleavage and polyadenylation

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Nuclear processing of RNA pol II transcripts
Nelson Cox, 2005, p. 1007
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Identification of introns by R-looping
Nelson Cox, 2000, p. 992
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Exonintron organization of the ovalbumin gene
Nelson Cox, 2000, p. 992
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General features of nuclear introns
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RNARNA interactions target snRNPs to 5-exon
junctions and internal branch site A residues
branch point
Nelson Cox, 2005, p. 1012
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Mechanism of nuclear splicing
adapted from Nelson Cox, 2005, p. 1012
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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
Alberts et al, 2002, p.318
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Mechanism of nuclear splicing
adapted from Nelson Cox, 2005, p. 1012
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Other types of introns

• 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
• tRNA introns
• endonucleases cleave RNA at both ends of the
  intron
• exons are ligated together after additional
  ATP-dependent modifications

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The mRNA 5-cap structure

• Functions
• marks the 5-end of the first exon and aids in
  the splicing process
• 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 processing of mRNA 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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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
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RNA processing mutations associated with
b-thalessemia
Alberts et al, 2002, p. 325
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rRNA processing in eukaryotes
Nelson Cox, 2005, p. 1016
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rRNA processing in prokaryotes
Nelson Cox, 2005, p. 1015
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tRNA processing
Nelson Cox, 2005, p. 1016
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Modified bases present in tRNAs
Nelson Cox, 2005, p. 1017
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Schematic of a mature mRNA
Wilson, 2003
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Mechanisms of cytoplasmic mRNA turnover
Wilson, 2003
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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
Features -internal guide sequence (yellow
box) -5 intronexon junction (red arrow) -3
intronexon junction (blue arrow) -complimentary
sequences near intronexon junctions (green
boxes)
Nelson Cox, 2005, p. 1018
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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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Nucleotidyl transfer activity of the L-19 IVS
Nelson Cox, 2005, p. 1019