Regulation of the Ysh1 endonuclease of the mRNA cleavage/polyadenylation complex by ubiquitin-mediated degradation.

Mutation of the essential yeast protein Ipa1 has previously been demonstrated to cause defects in pre-mRNA 3' end processing and growth, but the mechanism underlying these defects was not clear. In this study, we show that the ipa1-1 mutation causes a striking depletion of Ysh1, the evolutionarily conserved endonuclease subunit of the 19-subunit mRNA Cleavage/Polyadenylation (C/P) complex, but does not decrease other C/P subunits. YSH1 overexpression rescues both the growth and 3' end processing defects of the ipa1-1 mutant. YSH1 mRNA level is unchanged in ipa1-1 cells, and proteasome inactivation prevents Ysh1 loss and causes accumulation of ubiquitinated Ysh1. Ysh1 ubiquitination is mediated by the Ubc4 ubiquitin-conjugating enzyme and Mpe1, which in addition to its function in C/P, is also a RING ubiquitin ligase. In summary, Ipa1 affects mRNA processing by controlling the availability of the C/P endonuclease and may represent a regulatory mechanism that could be rapidly deployed to facilitate reprogramming of cellular responses.

Ipa1 Is an RNA Polymerase II Elongation Factor that Facilitates Termination by Maintaining Levels of the Poly(A) Site Endonuclease Ysh1.

The yeast protein Ipa1 was recently discovered to interact with the Ysh1 endonuclease of the pre-mRNA cleavage and polyadenylation (C/P) machinery, and Ipa1 mutation impairs 3'end processing. We report that Ipa1 globally promotes proper transcription termination and poly(A) site selection, but with variable effects on genes depending upon the specific configurations of polyadenylation signals. Our findings suggest that the role of Ipa1 in termination is mediated through interaction with Ysh1, since Ipa1 mutation leads to decrease in Ysh1 and poor recruitment of the C/P complex to a transcribed gene. The Ipa1 association with transcriptionally active chromatin resembles that of elongation factors, and the mutant shows defective Pol II elongation kinetics in vivo. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect, but not the mutant's sensitivity to 6-azauracil, an indicator of defective elongation. Our findings support a model in which an Ipa1/Ysh1 complex helps coordinate transcription elongation and 3' end processing.

The C terminus of Pcf11 forms a novel zinc-finger structure that plays an essential role in mRNA 3'-end processing.

3'-End processing of pre-mRNAs prior to packaging and export to the cytoplasm of the mature transcript is a highly regulated process executed by several tens of protein factors that recognize poorly conserved RNA signals. Among them is Pcf11, a highly conserved, multidomain protein that links transcriptional elongation, 3'-end processing, and transcription termination. Here we report the structure and biochemical function of Pcf11's C-terminal domain, which is conserved from yeast to humans. We identify a novel zinc-finger fold, resembling a trillium flower. Structural, biochemical, and genetic analyses reveal a highly conserved surface that plays a critical role in both cleavage and polyadenylation. These findings provide further insight into this important protein and its multiple functional roles during cotranscriptional RNA processing.

A global genetic interaction network maps a wiring diagram of cellular function.

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 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.

Efficient mRNA polyadenylation requires a ubiquitin-like domain, a zinc knuckle, and a RING finger domain, all contained in the Mpe1 protein.

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.

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3' end processing complex CF I.

The accuracy of the 3'-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of hetero-dimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3'-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.

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.

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition.

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

Analysis of interactions between mismatch repair initiation factors and the replication processivity factor PCNA.

In eukaryotes, the DNA replication factor PCNA is loaded onto primer-template junctions to act as a processivity factor for DNA polymerases. Genetic and biochemical studies suggest that PCNA also functions in early steps in mismatch repair (MMR) to facilitate the repair of misincorporation errors generated during DNA replication. These studies have shown that PCNA interacts directly with several MMR components, including MSH3, MSH6, MLH1, and EXO1. At present, little is known about how these interactions contribute to the mismatch repair mechanism. The interaction between MLH1 and PCNA is of particular interest because MLH1-PMS1 is thought to act as a matchmaker to signal mismatch recognition to downstream repair events; in addition, PCNA has been hypothesized to act in strand discrimination steps in MMR. Here, we utilized both genetic and surface plasmon resonance techniques to characterize the MLH1-PMS1-PCNA interaction. These analyses enabled us to determine the stability of the complex (K(D) = 300 nM) and to identify residues (572-579) in MLH1 and PCNA (126,128) that appear important to maintain this stability. We favor a model in which PCNA acts as a scaffold for consecutive protein-protein interactions that allow for the coordination of MMR steps.

cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four.

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 approximately 10 kb or until encountering competition from a euchromatic determinant.

DNA bending and unbending by MutS govern mismatch recognition and specificity.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.