1. Volume 12, Issue 6, Pages 1439-1452 (December 2003)
Genome-Wide Analysis of mRNAs Regulated by the Nonsense-Mediated and 5′ to 3′ mRNA Decay Pathways in Yeast 
Feng He, Xiangrui Li, Phyllis Spatrick, Ryan Casillo, Shuyun Dong, Allan Jacobson 
Molecular Cell 
Volume 12, Issue 6, Pages (December 2003)
DOI: /S (03)

2. Figure 1 Clustering Analysis of Differentially Expressed RNAs
(A) Expression patterns of five strains defective in either NMD or 5′ to 3′ mRNA decay. Twenty-six expression profiling experiments were carried out for the wild-type (wt), upf1Δ, nmd2Δ, upf3Δ, dcp1Δ, and xrn1Δ strains. A set of 2152 transcripts represented by 2271 probe sets exhibited more than 2-fold increases or decreases in their levels of expression and a change p value ≤0.05 in at least one of the mutant strains. These 2152 transcripts were clustered by using a hierarchical clustering algorithm (GeneSpring) in two dimensions according to gene and experiment vectors. In this analysis, the average difference of each transcript in the mutant strains was normalized to that in the wild-type strain. The resulting expression ratios from at least four independent replicates were averaged, transformed to base two logarithms, and clustered using the standard correlation coefficient as a distance metric. Genes are plotted along the horizontal axis, with the gene cluster tree above. Strains are plotted along the vertical axis, with the experiment cluster tree on the left side of the plot.
(B) NMD substrates are regulated by the 5′ to 3′ pathway even in the absence of NMD function. Twenty expression profiling experiments were carried out for the wild-type (wt), xrn1Δ, xrn1Δupf1Δ, xrn1Δnmd2Δ, and xrn1Δupf3Δ strains. A set of 1952 transcripts, represented by 2063 probe sets, exhibited more than 2-fold increases or decreases in their levels of expression and a change p value ≤0.05 in at least one of the mutant strains. These 1952 transcripts were clustered as described for (A).
In (A) and (B), color coding is used to represent the fold change in expression, as indicated within the inset color bar. Red indicates transcripts with increased expression levels in the mutant strains relative to the wild-type strain, and blue indicates transcripts with reduced expression levels.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 UPF1, NMD2, and UPF3 Regulate the Same Sets of Transcripts
(A–C) Scatter plots comparing the expression ratios of 1184 probe sets that exhibited more than 2-fold increases or decreases and a change p value ≤0.05 in the upf1Δ, nmd2Δ, or upf3Δ strains. The average difference for each transcript in the mutant strains was normalized to that in the wild-type strain. The resulting expression ratios from four independent replicates were averaged and plotted on a logarithmic scale for pair-wise comparisons. The dark line indicates the line of equivalence. The light lines indicate a 2-fold difference in expression. A spreadsheet listing the data for these 1184 probe sets is provided with the supplemental data.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Expression Patterns of Transcripts Regulated by NMD or the Components of the 5′ to 3′ Decay Pathway
(A and C) Venn diagrams summarizing the 1791 probe sets that exhibited more than 2-fold increases and a change p value ≤0.05 and the 306 probe sets that exhibited more than 2-fold decreases and a change p value ≤0.05 in the nmdΔ, dcp1Δ, or xrn1Δ strains, respectively.
(B and D) Self-organizing map analysis of the upregulated 1791 and the downregulated 306 probe sets in the nmdΔ, dcp1Δ, or xrn1Δ strains. The average difference of each transcript in the mutant strains was normalized to that in the wild-type strain. The resulting expression ratios from at least four independent replicates were averaged, transformed to base two logarithms, normalized by standardizing each row (gene) to mean = 0 and variance = 1. Transcripts were then grouped into 15 clusters (a 3 × 5 SOM). For each cluster, the y axis represents the relative expression levels and the horizontal axis represents different yeast strains in the order (from left to right): 1-wild-type, 2-upf1Δ, 3-nmd2Δ, 4-upf3Δ, 5-dcp1Δ, and 6-xrn1Δ. The mean expression level of each clustered strain is represented in blue and standard deviations are in red. The probe set number in each cluster is indicated. Spreadsheets listing the data for these 1791 and 306 probe sets are provided with the supplemental data.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Atypical Abundance and Chromosomal Location of Regulated Transcripts
(A) Transcripts upregulated in the nmdΔ, dcp1Δ, or xrn1Δ strains exhibit intrinsically low abundance in wild-type cells. Histograms are used to display the normal distributions of abundance (i.e., average difference) for the 7049 probe sets considered to be expressed in at least one of the six yeast strains analyzed (blue bars), the 792 probe sets upregulated in the nmdΔ strains (pink bars), the 1233 probe sets upregulated in the dcp1Δ strain (cyan bars), and the 1280 probe sets upregulated in the xrn1Δ strain (yellow bars). The horizontal axis represents the log10 transformed average difference values (see Experimental Procedures). The vertical axes represent the frequency of occurrence, with the left axis used for the data from all 7049 probe sets and the right axis used for the upregulated probe sets. In all cases, the average difference value for each transcript was the average of the values from five independent replicates carried out for the wild-type strain.
(B) Genome-wide map of the physical locations of genes upregulated in nmdΔ strains. Inactivation of the NMD pathway results in upregulation of 545 protein-coding genes. These genes were mapped to their respective chromosomal loci with horizontal green lines representing chromosomes and vertical red bars representing genes.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Validation of Microarray Data by Analysis of Individual RNAs
(A) Analysis of mRNA steady-state levels. (B) mRNA half-life measurements. (C) Analysis of poly(A) tail lengths. Total RNA was isolated from the designated strains or time points and characterized by Northern blotting (A and B), or by Northern blotting preceded by RNaseH digestion (in the presence [+] or absence [−] of oligo(dT); [C]). Multiple bands in some parts of (A) reflect either genomic homologs (e.g., THI11, GTT2, and YDLWTAU1) or RNA species with multiple 5′ or 3′ ends (e.g., MSS1 and YIL164C/165C).
Molecular Cell  , DOI: ( /S (03) )