Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approximately 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less ␤-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.In Saccharomyces cerevisiae RNA polymerase II (RNAPII) initiates transcription in concert with general transcription factors and a 20-subunit mediator complex (16,38,59). During initiation it becomes phosphorylated by the Kin28 subunit of the general transcription factor TFIIH on Ser5 of the heptapeptide repeats, YSPTSPS, in the carboxy-terminal domain (CTD) of its largest subunit, Rpb1, resulting in recruitment of the mRNA-capping enzyme to the transcription complex (23,32,49,69). Ser5 phosphorylation declines during the early stages of elongation and is replaced by Ser2 phosphorylation, mediated mainly by the cyclin-dependent kinase, Ctk1, during the later stages of elongation by RNAPII (10, 23). General transcription factors and mediator dissociate from the transcription complex or remain behind at the promoter and are replaced by various elongation factors during chain elongation by RNAPII (25,43). Among these elongation factors are Spt4/ Spt5, Spt6/Iws1, and Spt16/Pob3, whose patterns of genetic interactions suggest that they participate in transcription on chromatin templates (66). Another elongation factor is the Paf1 complex, which consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and associates with RNAPII (25, 34, 52). Chromatin immunoprecipitation (ChIP) experiments have shown that all of these polypeptides are associated with transcribed regions (25,43).Histone methylation by SET domain-containing histone lysine methyltransferases has important roles in chromatin structure and function (44). Lysines 4, 9, 27, and 79 are well-studied sites of methylation on histone H3, while lysine 20 is the only known methylated lysine in histone H4 (55, 64). The...

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.