Precision and functional specificity in mRNA decay

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Precision and functional specificity in mRNA
decay

• Yulei Wang , Chih Long Liu , John D. Storey
• PNAS, vol 99, NO.9, 2002

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Purposeto learn the specificity, precision and
regulatory role of mRNA decay
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  Materials and Methods

• Yeast Strain
• Saccharomyces cerevisiae strain Y262 (MATa
  ura3-52 his4-939am rpb1-1), carrying a
  temperature-sensitive mutation in RNA polymerase
  II, was used in this study.

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Determination of mRNA Decay by Transcriptional
Shut-Off AssayY262 was grown in 500 ml of yeast
extract/peptone/dextrose (YPD) medium at 24C to
OD600 0.5. The temperature of the culture was
abruptly shifted to 37C by adding an equal
volume of YPD medium that had been prewarmed to
49C. Aliquots of the culture (100 ml) were
removed at 0, 5, 10, 15, 20, 30, 40, 50, and
60 min after the temperature shift. Cells were
rapidly harvested on a nitrocellulose filter
(Whatman no. 141109) followed by immediate
freezing in liquid N2. Total RNA was prepared
from cells harvested at each time point by hot
phenol extraction.
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Microarray Analysis

• For total RNA sample (15-75 µg) at each time
  point, a fluorescently labeled cDNA probe was
  prepared by reverse transcription in the presence
  of Cy5-dUTP, using a random primer
• Yeast genomic DNA (200 ng) was digested with
  DpnII (New England Biolabs) into 0.3-2-kb
  fragments, labeled with Cy3-dUTP by using reverse
  transcriptase

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Statistical Analysis of mRNA Decay in
Stoichiometric Protein Complexes.
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Results

• Whole-Genome Determination of mRNA Half-Lives.

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(No Transcript)
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Determination of Poly(A) Shortening Ratesusing
an anchored oligo(dT) primer (5'-T20VN-3'),
rather than random primers, in the cDNA probe
synthesis. This approach allowed us to track
specifically the mRNAs that retained poly(A)
tails of sufficient length to allow priming.
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• Comparison between overall mRNA decay rates and
  poly(A) mRNA decay rates. (A) Distribution of
  half-lives of mRNA overall decay (blue) and
  poly(A) mRNA decay (red). (B) Scatter plot of
  half-lives of mRNA overall decay and poly(A)
  mRNA decay for the 4,661 mRNAs. Cor.
  Coeff.  0.50. The pink dashed line indicates a
  slope of 1. The green solid line is the best
  least-squares linear fit of the data, with a
  slope of 0.41 and y intercept of 1 min (in log
  scale).

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Precision in mRNA Decay
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• (A) Examples of coordinated decay of transcripts
  for four stoichiometric protein complexes. The
  number of unique components in each complex is
  indicated in parentheses

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• Clustering of the decay half-lives of mRNAs
  encoding subunits of protein complexes.

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Physiological Coherence in mRNA Decay
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• Coherence of mRNA turnover in physiological
  systems. The range of half-lives, from 0 to more
  than 45 min, was continuously color-coded with a
  green-yellow-red gradient (green  shortest
  half-lives, red  longest half-lives). For
  protein complexes with multiple subunits, smaller
  blocks were individually color-coded to represent
  the mRNA half-lives for each subunit. White boxes
  represent transcripts for which we did not obtain
  an adequate measurement of decay. TCA,
  tricarboxylic acid. mRNA turnover in (A) central
  energy metabolism systems

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• (B) the pheromone signal transduction pathway

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• (C) translation factors.

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Good Night