A SUPPLEMENT FOR

L. Kuai and F. Sherman, Genome-wide Analysis of Half-lives of mRNAs from Saccharomyces cerevisiae (in preparation).

Table 1

Table 2

L. Kuai, B. Das and F. Sherman. The nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. (in press) (2005).

Table 3

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Table 1 contains and compares the half-lives of mRNAs of a total of 2,379 genes that are common to the data sets of Wang et al. (2002) (designated Wang), Grigull et al. (2004) (designated Grigull), and Kuai and Sherman (in preparation) (designated This Study). Only half-lives between 0-80 mins are presented. The genes showing half-life ratio between 0.5 and 2 were considered "agreed" between data sets and were assigned a score of 1. The genes with fold change greater than 2 were considered "disagreed" and were assigned a score of 0.

"Gene Name" refers to the systematic name of the gene. "Symbol" refers to the standard name of the gene (http://www.yeastgenome.org). All half-lives in minute were rounded to integers. The following three comparisons were made: G/W (Grigull and Wang); W/K (Wang and This Study); and G/K (Grigull and This Study). R2 designates the correlation coefficient of linear fits of our data.

Table 2 contains the half-lives of mRNAs of a total of 9,335 probe sets and corresponding gene half-life values from Grigull et al. (2004) supplementary Table 2, which includes half-lives previously reported in Wang et al. (2002). All half-lives in minute were rounded to integers.

"Probe Name"refers to the probe ID assigned to each probe set in Affymetrix S98 GeneChipÒ (www.affymetrix.com). "Gene Name", "Symbol", "R2", "Grigull", "Wang" and "This study"are defined above in the Table 1 description.

Table 3 contains the relative steady state levels and half-lives of mRNAs from normal and cbc1-D, rrp6-D and upf1-D deletion strains of Saccharomyces cerevisiae.

The steady state mRNA levels were determined by global normalization of the time 0 data of the Normal, cbc1-D, rrp6-D and upf1-D strains, using the normal strain as baseline. The normalization was performed by Affymetrix MAS 5.0 data processing software. A detection call (CALL) was assigned to each value as follows: "P", present (detected signal intensity greater than threshold); "A", Absent (detected signal intensity less than threshold); "M", Marginal (detected signal close to threshold). The signal intensity of times 0, 5 and 20 min were normalized with the specialized program HLeditor 0.01 by assuming that the ACT1 mRNA half-life is 64 min. The Half-life in minutes of each ORF was obtained by least square regression of the natural log of signal intensities at the three time points. The correlation coefficient R² of each linear fitting was recorded as the quality of the linear fitting. Negative values of half-life were represented as "N.A." (not applicable). Only the 6,872 probe sets representing the Open Reading Frames (GO annotation, http://www.geneontology.org/GO.annotation.html) are listed.

Microarray Sample Handling and Data Processing. Cells were grown in YPD medium to A_{600 nm}=1.0 followed by treatment of 4 mg/ml thiolutin. Cells were collected at the following three time-points: before treatment, 5 min after treatment and 20 min after treatment. Total RNA was isolated using the method described above and labeled by 2-rounds of in vitro transcription with T7-Oligo(dT) Promoter Primer Kit (Affymetrix^®), following the procedure recommended by the manufacturer. The fluorescent-labeled cRNA was hybridized to the Affymetrix^® Yeast S98 Gene chip and scanned by Hewlett Packard^® GeneArray Scanner. The fluorescent signals corresponding to hybridization intensities were analyzed with Affymetrix^® Microarray Suite software (version 5.0, www.affymetrix.com). The chips, hybridized to the time 0 sample of each strain, were normalized with global scaling normalization to conduct the steady state level of each transcript. The software substrates the signal for each mismatched oligonucleotide (MM) from the signal for each perfectly matched oligonucleotide (PM). The adjusted signals for each probe pair (PM-MM) are averaged across each probe pair set, excluding the probe pairs that give signals 3 standard deviations or more from the mean. A present call "P", marginal call "M" or absent call "A" was assigned to each probe set representing the ratio between signal reported and the detection threshold. For half-life determination, the data from 3 time points were normalized so that the half-life of ACT1 mRNA is 50 min and the normalizations factors were applied to the whole data sets.