Claire Moore

RNA-binding proteins: modular design for efficient function

Many RNA-binding proteins have modular structures and are composed of multiple repeats of just a few basic domains that are arranged in various ways to satisfy their diverse functional requirements. Recent studies have investigated how different modules cooperate in regulating the RNA-binding specificity and the biological activity of these proteins. They have also investigated how multiple modules cooperate with enzymatic domains to regulate the catalytic activity of enzymes that act on RNA. These studies have shown how, for many RNA-binding proteins, multiple modules define the fundamental structural unit that is responsible for biological function.

Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

ABSTRACT The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase–CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, whilesrb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

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

INTRODUCTION Genetic interactions occur when mutations in two or more genes combine to generate an unexpected phenotype. An extreme negative or synthetic lethal genetic interaction occurs when two mutations, neither lethal individually, combine to cause cell death. Conversely, positive genetic interactions occur when two mutations produce a phenotype that is less severe than expected. Genetic interactions identify functional relationships between genes and can be harnessed for biological discovery and therapeutic target identification. They may also explain a considerable component of the undiscovered genetics associated with human diseases. Here, we describe construction and analysis of a comprehensive genetic interaction network for a eukaryotic cell. RATIONALE Genome sequencing projects are providing an unprecedented view of genetic variation. However, our ability to interpret genetic information to predict inherited phenotypes remains limited, in large part due to the extensive buffering of genomes, making most individual eukaryotic genes dispensable for life. To explore the extent to which genetic interactions reveal cellular function and contribute to complex phenotypes, and to discover the general principles of genetic networks, we used automated yeast genetics to construct a global genetic interaction network. RESULTS We tested most of the ~6000 genes in the yeast Saccharomyces cerevisiae for all possible pairwise genetic interactions, identifying nearly 1 million interactions, including ~550,000 negative and ~350,000 positive interactions, spanning ~90% of all yeast genes. Essential genes were network hubs, displaying five times as many interactions as nonessential genes. The set of genetic interactions or the genetic interaction profile for a gene provides a quantitative measure of function, and a global network based on genetic interaction profile similarity revealed a hierarchy of modules reflecting the functional architecture of a cell. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections associated with defects in cell cycle progression or cellular proteostasis. Importantly, the global network illustrates how coherent sets of negative or positive genetic interactions connect protein complex and pathways to map a functional wiring diagram of the cell. CONCLUSION A global genetic interaction network highlights the functional organization of a cell and provides a resource for predicting gene and pathway function. This network emphasizes the prevalence of genetic interactions and their potential to compound phenotypes associated with single mutations. Negative genetic interactions tend to connect functionally related genes and thus may be predicted using alternative functional information. Although less functionally informative, positive interactions may provide insights into general mechanisms of genetic suppression or resiliency. We anticipate that the ordered topology of the global genetic network, in which genetic interactions connect coherently within and between protein complexes and pathways, may be exploited to decipher genotype-to-phenotype relationships. A global network of genetic interaction profile similarities. (Left) Genes with similar genetic interaction profiles are connected in a global network, such that genes exhibiting more similar profiles are located closer to each other, whereas genes with less similar profiles are positioned farther apart. (Right) Spatial analysis of functional enrichment was used to identify and color network regions enriched for similar Gene Ontology bioprocess terms. 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.

Unravelling the means to an end: RNA polymerase II transcription termination

The pervasiveness of RNA synthesis in eukaryotes is largely the result of RNA polymerase II (Pol II)-mediated transcription, and termination of its activity is necessary to partition the genome and maintain the proper expression of neighbouring genes. Despite its ever-increasing biological significance, transcription termination remains one of the least understood processes in gene expression. However, recent mechanistic studies have revealed a striking convergence among several overlapping models of termination, including the poly(A)- and Sen1-dependent pathways, as well as new insights into the specificity of Pol II termination among its diverse gene targets. Broader knowledge of the role of Pol II carboxy-terminal domain phosphorylation in promoting alternative mechanisms of termination has also been gained.

Organization and Function of APT, a Subcomplex of the Yeast Cleavage and Polyadenylation Factor Involved in the Formation of mRNA and Small Nucleolar RNA 3′-Ends

Messenger RNA 3′-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

Coupling of Termination, 3 Processing, and mRNA Export

ABSTRACT In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3′ processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3′ processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3′ processed transcripts at the nonpermissive temperature. 3′ maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3′ processing. The similarity of the phenotypes of some mRNA export mutants and 3′ processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3′ processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3′ maturation. Of the core 3′ processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3′ processing and mRNA export defects found in xpo1-1 cells.

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Cleavage and polyadenylation of mRNA 3′ ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein–protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation

ABSTRACT In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3′ end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3′ end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.

Pta1, a component of yeast CF II, is required for both cleavage and poly(A) addition of mRNA precursor.

ABSTRACT CF II, a factor required for cleavage of the 3′ ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides. The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition. In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing. Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex. Extracts prepared frompta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1substrates and exhibit little processing activity even after prolonged incubation. However, activity is efficiently rescued by the addition of purified CF II to the defective extracts. Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not. The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated. Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I. While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo. The involvement of Pta1 in both steps of mRNA 3′-end formation supports the conclusion that CF II is the functional homolog of CPSF.