We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing over 23 million double mutants, identifying ~550,000 negative and ~350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for ϳ10 kb or until encountering competition from a euchromatic determinant.The chromosomes of higher eukaryotes are organized into multiple domains with distinct properties. The partitioning of chromosomes into zones of condensed heterochromatin and dispersed euchromatin is readily seen in interphase nuclei. Heterochromatin has the property of silencing most genes that are normally packaged in euchromatin; this silencing is strikingly evident where euchromatic genes are abnormally juxtaposed to heterochromatic domains by chromosome rearrangement or transposition, resulting in position-effect variegation (PEV) (16). Work with multiple systems has led to a model of heterochromatin formation based on a specific combination of biochemical marks. Heterochromatic regions are characterized by histone hypoacetylation and methylation of histone H3 at lysine 9 (producing H3-mK9), in most cases accompanied by binding of heterochromatin protein 1 (HP1) and in some cases associated with methylation of the DNA (31). In Drosophila melanogaster, mutations in HP1 [Su(var)2-5] and in an H3-K9 methyltransferase [Su(var)3-9] cause a loss of heterochromatin-induced silencing. A remarkable property of heterochromatin is the ability to spread in cis in response to the loss of boundary constraints or to changes in the dosage or activity of chromatin components (24,38). The interaction of HP1 with both the modified histone H3-mK9 and the modifying enzyme SU(VAR)3-9 suggests a plausible model for the maintenance and spreading of heterochromatin (reviewed in reference 16).

Almost all eukaryotic mRNAs must be polyadenylated at their 3= ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3=-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3=-end processing. Polyadenylation is an essential step in the production of functional eukaryotic mRNA that will be efficiently utilized in translation. It is a nuclear processing event that involves the cleavage of mRNA precursor followed by the addition of a poly(A) tail and is carefully coordinated with other events involved in mRNA synthesis and utilization, such as transcription, splicing, assembly of mRNA into ribonucleoprotein complexes, and mRNA export. Many recent studies have also revealed how selection of the cleavage site can globally affect the type of mRNA produced by cells (1-3). Polyadenylation requires a suite of multiple factors whose subunits are conserved across eukaryotic species (4-6). Significant progress has been made in understanding the contributions of each factor to the recognition of the poly(A) site, the execution of the processing steps, and regulation. However, the precise role of each subunit in this complex process and the impact of posttranslational modifications on the regulation of polyadenylation have not been completely defined.In Saccharomyces cerevisiae, the factors needed for mRNA 3=-end processing are the RNA-binding protein Hrp1 and two multisubunit complexes called cleavage/polyadenylation factor (CPF) and cleavage factor IA (CF IA). One poorly characterized but essential subunit of CPF identified more than 10 years ago is Mpe1, which is needed for both processing steps (7). The mammalian Mpe1 homolog, RBBP6, interacts with the tumor suppressor proteins p53 and pRb (8, 9), possibly linking polyadenylation to regulation of cell growth. Even though RBBP6 is found in the mammalian mRNA 3=-end processing complex (10), it has not been shown to function in polyadenylation.Mpe1 is a particularly interesting s...

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage–Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1–Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3′-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1–Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.