Precision and functional
specificity in mRNA decay
Yulei Wang , Chih Long Liu , John D. Storey
PNAS, vol 99, NO.9, 2002
Purpose
to learn the specificity, precision and
regulatory role of mRNA decay
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.
Determination of mRNA Decay by Transcriptional
Shut-Off Assay
Y262 was grown in 500 ml of yeast extract/peptone/dextrose (YPD)
medium at 24°C to OD600 ~0.5. The temperature of the culture
was abruptly shifted to 37°C by adding an equal volume of YPD
medium that had been prewarmed to 49°C. 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.
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
Statistical Analysis of mRNA
Decay in Stoichiometric Protein
Complexes.
Results

Whole-Genome Determination of mRNA HalfLives.
Determination of Poly(A)
Shortening Rates
using 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.

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).
Precision in mRNA Decay

(A) Examples of coordinated decay of transcripts for four
stoichiometric protein complexes. The number of unique
components in each complex is indicated in parentheses

Clustering of the decay half-lives of mRNAs encoding
subunits of protein complexes.
Physiological Coherence in mRNA
Decay

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

(B) the pheromone signal transduction
pathway

(C) translation factors.
Good Night