Gene Expression Profiling of <i>puf3</i> Mutants

<p>Distribution of average Cy5/Cy3 fluorescence ratios from three independent microarray hybridizations comparing mRNA levels of <i>puf3</i>Δ with wild-type cells grown in minimal media with glycerol. The left frequency axis refers to all genes (black line); the axis to the right refers to Puf3p and Puf4p (control) targets, shown as red and blue lines, respectively. Relative expression levels of the 220 Puf3p mRNA targets in <i>puf3</i>Δ cells were selectively increased compared to all other mRNAs analyzed (<i>p</i> < 10⁻³⁴), whereas Puf4p targets were not (<i>p</i> > 0.05). Thirty-nine genes involved in aerobic respiration (according to GO annotation and SGD), but not bound by Puf3p, were similarly enriched (<i>p</i> < 5 × 10⁻⁵) in the <i>puf3</i> mutant as random sets of 39 Puf3p targets (<i>p</i> < 10⁻⁶). Likewise, 220 randomly selected mRNAs coding for mitochondrial proteins that were not associated with Puf3p in the experiments herein were weakly enriched in the mutant (<i>p</i> < 10⁻⁸).</p

Classification of mRNAs Interacting with Puf Proteins

<div><p>(A) Column charts showing compartmentalization of characterized gene products encoded by the Puf targets. The same compartments are shown for the entire genome in the columns designed “All” (YPD, May 2003). The number of genes represented in the charts is indicated on the top of columns. An asterisk indicates classes with <i>p</i> values of less than 0.001.</p> <p>(B) Fraction of membrane-associated gene products among the Puf targets. We classified the targets by combining both GO and YPD annotations (May 2003). “Plasma membrane” (light blue) is a subpopulation of the total membrane-associated proteins (blue). Soluble cytoplasmic or nuclear proteins were classified as “non-membrane.” “All” refers to the genome-wide compartmentalization of characterized genes, and respective numbers were retrieved from YPD. “Puf2 Top 40” refers to the 40 highest enriched Puf2p targets and equals the total number of Puf1p targets.</p></div

Sequence Motifs Interacting with Puf Proteins

<div><p>(A) Consensus motifs detected within 3′-UTR sequences of Puf3p, Puf4p, and Puf5p target mRNAs. Height of the letters specifies the probability of appearing at the position in the motif. Letters with less than 10% appearance were omitted. Fraction of genes bearing a motif in the 3′-UTR sequence is indicated to the right. Y-helicase proteins are nearly identical in sequence and were excluded from this analysis.</p> <p>(B) Scheme of three-hybrid assay for monitoring RNA–protein interactions in vivo (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020079#pbio-0020079-Bernstein1" target="_blank">Bernstein et al. 2002</a>).</p> <p>(C) β-Galactosidase activity for three-hybrid assay. Proteins assayed are indicated on top, RNAs to the left. Abbreviations: pum, pum-HD; cons., consensus motif; UGU/AGA, UGU in consensus sequence mutated to AGA.</p> <p>(D) Activation of <i>HIS3</i> reporter gene and resistance to 3-aminotriazole (3-AT), a competitive inhibitor of the <i>HIS3</i> gene product, in a three-hybrid assay (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020079#pbio-0020079-Bernstein1" target="_blank">Bernstein et al. 2002</a>).</p></div

Defining Puf Target RNAs

<div><p>(A) Distribution of average Cy5/Cy3 fluorescence ratios from four independent microarray hybridizations analyzing Puf3p targets. The arrow depicts enrichment of <i>COX17</i> mRNA, which is known to bind to Puf3p (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020079#pbio-0020079-Olivas1" target="_blank">Olivas and Parker 2000</a>). The red dashed line indicates the threshold applied for defining 220 target RNAs (a magnification is shown of the enriched region).</p> <p>(B) Cluster of RNA targets for Puf proteins. Rows represent genes (unique cDNA elements) and columns represent individual experimental samples. Each Puf protein and an untagged strain (mock control) were assayed in quadruplicate. The color code indicates enrichments (green–red color scale). The number of mRNAs interacting with each Puf protein is indicated in parentheses. mRNAs clustering with the mock controls were removed as false positives (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020079#s4" target="_blank">Materials and Methods</a>).</p></div

Localization of Puf Proteins

<p>TAP-tagged Puf proteins were visualized in fixed cells. DNA was costained with 4′,6-diamidino-2-phenylindole dimethylsulfoxide (DAPI).</p

Strategy for Analyzing Genome-Wide RNA–Protein Interactions

<p>Protein A-tagged Puf proteins were captured with IgG–Sepharose and released from the beads by cleavage with TEV protease. RNAs associated with the released proteins were isolated, and cDNA copies were fluorescently labeled and hybridized to yeast DNA microarrays. The Cy5/Cy3 fluorescence ratio for each locus reflects its enrichment by affinity for the cognate protein.</p

Protein Domain Structure of Yeast Puf Proteins

<p>Pum-HD repeats (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020079#pbio-0020079-Zamore1" target="_blank">Zamore et al. 1997</a>) are shown as red ovals and classical RNA-binding domains (RBDs) are depicted as blue boxes. Regions of low complexity, such as proline-, serine-, threonine-, and/or methionine-rich domains, are shown in gray boxes; asparagine stretches are striped. The numbers correspond to the length of proteins in amino acids.</p

Specific Proteins Bind Functional Groups of Genes for Regulation

<p>At the transcriptional level (top), transcription factors (TFs) regulate initiation of transcription (green arrow) in the nucleus by binding to sequence elements (yellow box) proximal to their target coding regions (boxes). At the post-transcriptional level (middle), RBPs regulate decay, translation, or localization of mRNAs in a coordinated fashion by interaction with sequence/structural elements in the RNA that are often found in 3′-UTR regions (red box). Functional relations at the protein level (bottom) can be reflected at both the transcriptional and post-transcriptional levels: sets of genes that encode functionally related proteins, such as subunits of stochiometric complexes (blue) or components of the same regulatory or metabolic pathway (gray and cross-hatched boxes), may be regulated by common transcription factors and their mRNAs post-transcriptionally coregulated by RBPs (dashed interactions).</p

Identification of Markers of Organ-Specific Injury

<p>The top-20 CN array elements for each normal tissue were selected using a Student's <i>t</i>-test comparing each normal tissue to all other normal tissues. All normal tissues represented by at least five microarray experiments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020011#pgen-0020011-g004" target="_blank">Figure 4</a> were included (150 microarrays). Only array elements that passed data quality filters for at least 70% of all normal tissue experiments were considered. Data are displayed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020011#pgen-0020011-g004" target="_blank">Figure 4</a>, and the positions of several genes are indicated.</p

Large-Scale Categorization of MS and CN Genes

<p>We evaluated the ability of various array element-specific parameters to classify genes encoding MS (A) or CN (B) proteins, using receiver operator analysis. Based on these analyses, we chose the average log₂ Cy5/Cy3 ratio for assigning the final localization annotations (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020011#pgen-0020011-sd004" target="_blank">Protocol S1</a>). The curves were generated by incrementally relaxing the parameter cutoff values to generate gene sets with varying fractions of known MS- or CN-encoding genes. (C) Relationship between sensitivity, specificity, fraction of characterized genes encoding MS proteins, and the total number of clones classified as MS, using the average log₂ Cy5/Cy3 ratio as the selection criteria. The vertical arrows indicate two cutoffs used for subsequent analyses. (D) Same as in (C) but for genes encoding CN proteins.</p