Precision and functional spPrecision and functional specificity in mRNA decay ecificity in mRNA decay

Yulei Wang , Chih Long Liu , John D. Storey

PNAS, vol 99, NO.9, 2002

PurposePurposeto learn the specificity, precision and regto learn the specificity, precision and reg

ulatory role of mRNA decayulatory role of mRNA decay

    Materials and MethodsMaterials 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 SDetermination of mRNA Decay by Transcriptional Shut-Off Assayhut-Off Assay

Y262 was grown in 500 ml of yeast extract/peptone/dextrose (YPD) Y262 was grown in 500 ml of yeast extract/peptone/dextrose (YPD) medium at 24°C to ODmedium at 24°C to OD

600600 ~0.5. The temperature of the culture ~0.5. The temperature of the culture was was

abruptly shifted to 37°C by adding an equal volume of YPDabruptly shifted to 37°C by adding an equal volume of YPD medium medium that had been prewarmed to 49°C. Aliquots of the culturethat had been prewarmed to 49°C. Aliquots of the culture (100 ml) (100 ml) were removed at 0, 5, 10, 15, 20, 30, 40, 50, and 60were removed at 0, 5, 10, 15, 20, 30, 40, 50, and 60   min after the temin after the temperature shift. Cells were rapidly harvestedmperature shift. Cells were rapidly harvested on a nitrocellulose filton a nitrocellulose filter (Whatman no. 141109) followed by immediateer (Whatman no. 141109) followed by immediate freezing in liquid freezing in liquid NN22. Total RNA was prepared from cells harvested. Total RNA was prepared from cells harvested at each time point at each time point

by hot phenol extraction. by hot phenol extraction.

Microarray AnalysisMicroarray 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 DecaStatistical Analysis of mRNA Decay in Stoichiometric Protein Comply in Stoichiometric Protein Compl

exes.exes.

Results Results

Whole-Genome Determination of mRNA Half-Lives.

Determination of Poly(A) ShorteniDetermination of Poly(A) Shortening Ratesng Rates

using an anchored oligo(dT) primer (5'-Tusing an anchored oligo(dT) primer (5'-T2020VN-3'),VN-3'), rather rather

than random primers, in the cDNA probe synthesis. Thisthan random primers, in the cDNA probe synthesis. This

approach allowed us to track specifically the mRNAs thaapproach allowed us to track specifically the mRNAs that retainedt retained poly(A) tails of sufficient length to allow priminpoly(A) tails of sufficient length to allow priming.g.

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 DecayPrecision 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 Physiological Coherence in mRNA DecayDecay

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