Assigning Function to Yeast Proteins by Integration of Technologies

Abstract

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…The protein seems to be neutral (pI = 7.31) and probably exists in low intracellular abundance on the basis of a codon adaptation index of 0.158. A database search revealed no obvious protein motifs for Npa1p, but its first 693 amino acids seem to have a predicted structure related to proteins of the HEAT-repeat family such as the PR65A subunit of protein phosphatase 2A (Yeast Resource Center Informatics Platform, http:// www.yeastrc.org; Hazbun et al 2003) and a low homology with Tpd3p, the yeast PR65A protein. We could also identify potential homologs of yeast Npa1p in human, Drosophila, Arabidopsis, and other fungi (Sequence Similarity Query Tool, Saccharomyces Genome Database; Christie et al 2004).…”

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

“…Eleven unannotated genes gave rise to G1 arrest after promoter shut-off, suggesting they might be involved in some aspect of ribosome biogenesis or protein biosynthesis. Indeed, high-throughput data suggest several of these genes perform such functions: YPR169W encodes a nucleolar protein of unknown function, which interacts with Tpt1p, a protein involved in tRNA splicing (Hazbun et al, 2003;Huh et al, 2003); Ynl313cp is localized to both cytoplasm and nucleus and interacts with proteins involved in RNA processing, ribosomal biogenesis import, and protein metabolism (Gavin et al, 2002;Huh et al, 2003;Krogan et al, 2004); Ynl310cp encodes a protein that interacts with proteins involved in RNA processing (Gavin et al, 2002;Ho et al, 2002;Hazbun et al, 2003); and Yhr020wp is inferred to have tRNA aminoacylation and ligation activity and also affinity-precipitates with Utp13, which is part of the SSU processome involved in pre-18S rRNA processing (Tatusov et al, 2000;Ho et al, 2002). Finally, Ynr046wp has been recently identified as subunit of tRNA methyltransferase (Purushothaman et al, 2005), and Ynr054cp, which encodes a nucleolar protein that interacts with proteins involved in RNA processing, has been recently characterized as a component of the SSU processome (Gavin et al, 2002;Ho et al, 2002;Huh et al, 2003;Hoang et al, 2005).…”

A pooling-deconvolution strategy for biological network elucidation

“…Thirteen of these bait strains have previously been screened against the genome-wide array. Because of experimental variability, these single bait screens required each bait to be screened in duplicate, resulting in a total of 32 screens for the 16 baits 2,14 . For the PI-Deconvolution format, the 16 bait strains were mixed into 8 pools and screened against the two-hybrid array.…”

“…In the 13 single bait screens, 484 preys were observed and defined as two-hybrid positive colonies 2,14 . Among these positive colonies, 125 arose twice out of the duplicate screens and were termed "reproducible" positives; the other 359 arose once as either false positives, or true positives that due to experimental variability did not yield reproducible results.…”