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1.  Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair 
PLoS Genetics  2009;5(2):e1000364.
RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.
Author Summary
Dealing with DNA lesions is one of the most important tasks of both prokaryotic and eukaryotic cells. This is particularly relevant for damage occurring inside genes, in the DNA strands that are actively transcribed, because transcription cannot proceed through a damaged site and the persisting lesion can cause either genome instability or cell death. Cells have evolved specific mechanisms to repair these DNA lesions, the malfunction of which leads to severe genetic syndromes in humans. Despite many years of intensive research, the mechanisms underlying transcription-coupled repair is still poorly understood. To gain insight into this phenomenon, we undertook a genome-wide screening in the model eukaryotic organism Saccharomyces cerevisiae for genes that affect this type of repair that is coupled to transcription. Our study has permitted us to identify and demonstrate new roles in DNA repair for factors with a previously known function in transcription, opening new perspectives for the understanding of DNA repair and its functional interconnection with transcription.
PMCID: PMC2629578  PMID: 19197357
2.  Yeast RNA Polymerase II Transcription In Vitro Is Inhibited in the Presence of Nucleotide Excision Repair: Complementation of Inhibition by Holo-TFIIH and Requirement for RAD26 
Molecular and Cellular Biology  1998;18(5):2668-2676.
The Saccharomyces cerevisiae transcription factor IIH (TFIIH) is essential both for transcription by RNA polymerase II (RNAP II) and for nucleotide excision repair (NER) of damaged DNA. We have established cell extracts which support RNAP II transcription from the yeast CYC1 promoter or NER of transcriptionally silent damaged DNA on independent plasmid templates and substrates. When plasmid templates and substrates for both processes are simultaneously incubated with these extracts, transcription is significantly inhibited. This inhibition is strictly dependent on active NER and can be complemented with purified holo-TFIIH. These results suggest that in the presence of active NER, TFIIH is preferentially mobilized from the basal transcription machinery for use in NER. Inhibition of transcription in the presence of active NER requires the RAD26 gene, the yeast homolog of the human Cockayne syndrome group B gene (CSB).
PMCID: PMC110646  PMID: 9566886
3.  Functional analysis of archaeal MBF1 by complementation studies in yeast 
Biology Direct  2011;6:18.
Multiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMbf1 were performed.
In contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1.
However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus SCM1.
The absence of MBF1-interacting activators in the archaeal domain, the presence of a Zn ribbon motif in the divergent N-terminal domain of aMBF1 and the complementation experiments using archaeal- yeast chimeric proteins presented here suggests that archaeal MBF1 is not able to functionally interact with the transcription machinery and/or Gcn4 of S. cerevisiae. Based on modeling and structural prediction it is tempting to speculate that aMBF1 might act as a single regulator or non-essential transcription factor, which directly interacts with DNA via the positive charged linker or the basal transcription machinery via its Zn ribbon motif and the HTH domain. However, also alternative functions in ribosome biosynthesis and/or functionality have been discussed and therefore further experiments are required to unravel the function of MBF1 in Archaea.
This article was reviewed by William Martin, Patrick Forterre, John van der Oost and Fabian Blombach (nominated by Eugene V Koonin (United States)). For the full reviews, please go to the Reviewer's Reports section.
PMCID: PMC3062615  PMID: 21392374
4.  Human Cytomegalovirus pUL79 Is an Elongation Factor of RNA Polymerase II for Viral Gene Transcription 
PLoS Pathogens  2014;10(8):e1004350.
In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection.
Author Summary
In this study, we report a novel mechanism used by human cytomegalovirus (HCMV) to regulate the elongation rate of RNA polymerase II (RNAP II) to facilitate viral transcription during late stages of infection. Recently, we and others have identified several viral factors that regulate gene expression during late infection. These factors are functionally conserved among beta- and gamma- herpesviruses, suggesting a unique transcriptional regulation shared by viruses of these two subfamilies. However, the mechanism remains elusive. Here we show that HCMV pUL79, one of these factors, interacts with RNAP II as well as other viral factors involved in late gene expression. We have started to elucidate the nature of the pUL79-RNAP II interaction, finding that pUL79 does not alter the protein levels of RNAP II or its recruitment to viral promoters. However, during late times of infection, pUL79 helps RNAP II efficiently elongate along the viral DNA template to transcribe HCMV genes. Host genes are not regulated by this pUL79-mediated mechanism. Therefore, our study discovers a previously uncharacterized mechanism where RNAP II activity is modulated by viral factor pUL79, and potentially other viral factors as well, for coordinated viral transcription.
PMCID: PMC4148446  PMID: 25166009
5.  In TFIIH, XPD Helicase Is Exclusively Devoted to DNA Repair 
PLoS Biology  2014;12(9):e1001954.
The DNA helicase activity of the xeroderma pigmentosum D protein, a crucial subunit of TFIID, is only needed for its role in DNA repair, not for transcription.
The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.
Author Summary
The multiprotein complex TFIIH is crucially involved in two fundamental cellular processes—the transcription of genes by RNA polymerase II and the repair of UV-induced DNA damage by a mechanism called nucleotide excision repair (NER). The xeroderma pigmentosum complementation group D (XPD) helicase, which is mutated in the eponymous human photosensitivity and cancer syndrome, is an important subunit of TFIIH, where it is assumed to act as a helicase, unwinding the DNA double helix. In our study, we show that XPD assumes an entirely different role in transcription and NER. In the case of repair, this protein works as an enzyme, requiring all its known functional properties. For transcription, however, none of the enzymatic functions is essential and XPD switches from an enzyme to a structural protein whose job is merely to preserve the integrity of the TFIIH complex. We have shown thus that the helicase activity of the XPD protein is exclusively devoted to repair processes and could be targeted by drugs without affecting transcription.
PMCID: PMC4182028  PMID: 25268380
6.  Comparative genomics of cyclin-dependent kinases suggest co-evolution of the RNAP II C-terminal domain and CTD-directed CDKs 
BMC Genomics  2004;5:69.
Cyclin-dependent kinases (CDKs) are a large family of proteins that function in a variety of key regulatory pathways in eukaryotic cells, including control over the cell cycle and gene transcription. Among the most important and broadly studied of these roles is reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II, part of a complex array of CTD/protein interactions that coordinate the RNAP II transcription cycle. The RNAP CTD is strongly conserved in some groups of eukaryotes, but highly degenerate or absent in others; the reasons for these differences in stabilizing selection on CTD structure are not clear. Given the importance of reversible phosphorylation for CTD-based transcription, the distribution and evolutionary history of CDKs may be a key to understanding differences in constraints on CTD structure; however, the origins and evolutionary relationships of CTD kinases have not been investigated thoroughly. Moreover, although the functions of most CDKs are reasonably well studied in mammals and yeasts, very little is known from most other eukaryotes.
Here we identify 123 CDK family members from animals, plants, yeasts, and four protists from which genome sequences have been completed, and 10 additional CDKs from incomplete genome sequences of organisms with known CTD sequences. Comparative genomic and phylogenetic analyses suggest that cell-cycle CDKs are present in all organisms sampled in this study. In contrast, no clear orthologs of transcription-related CDKs are identified in the most putatively ancestral eukaryotes, Trypanosoma or Giardia. Kinases involved in CTD phosphorylation, CDK7, CDK8 and CDK9, all are recovered as well-supported and distinct orthologous families, but their relationships to each other and other CDKs are not well-resolved. Significantly, clear orthologs of CDK7 and CDK8 are restricted to only those organisms belonging to groups in which the RNAP II CTD is strongly conserved.
The apparent origins of CDK7 and CDK8, or at least their conservation as clearly recognizable orthologous families, correlate with strong stabilizing selection on RNAP II CTD structure. This suggests co-evolution of the CTD and these CTD-directed CDKs. This observation is consistent with the hypothesis that CDK7 and CDK8 originated at about the same time that the CTD was canalized as the staging platform RNAP II transcription. Alternatively, extensive CTD phosphorylation may occur in only a subset of eukaryotes and, when present, this interaction results in greater stabilizing selection on both CTD and CDK sequences. Overall, our results suggest that transcription-related kinases originated after cell-cycle related CDKs, and became more evolutionarily and functionally diverse as transcriptional complexity increased.
PMCID: PMC521075  PMID: 15380029
7.  Tfb5 Is Partially Dispensable for Rad26 Mediated Transcription Coupled Nucleotide Excision Repair in Yeast 
DNA repair  2007;6(11):1661-1669.
Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of an actively transcribed gene. To be distinguished from TCR-NER, the genome-wide NER process is termed as global genomic NER (GG-NER). In Saccharomyces cerevisiae, GG-NER is dependent on Rad7, whereas TC-NER is mediated by Rad26, the homolog of the human Cockayne syndrome group B protein, and by Rpb9, a nonessential subunit of RNA polymerase II. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy. Here, we show that Tfb5 plays different roles in different NER pathways in yeast. No repair takes place in the nontranscribed strand of a gene in tfb5 cells, or in both strands of a gene in rad26 rpb9 tfb5 cells, indicating that Tfb5 is essential for GG-NER. However, residual repair occurs in the transcribed strand of a gene in tfb5 cells, suggesting that Tfb5 is important, but not absolutely required for TC-NER. Interestingly, substantial repair occurs in the transcribed strand of a gene in rad7 tfb5 and rad7 rpb9 tfb5 cells, indicating that, in the absence of GG-NER, Tfb5 is largely dispensable for Rad26 mediated TC-NER. Furthermore, we show that no repair takes place in the transcribed strand of a gene in rad7 rad26 tfb5 cells, suggesting that Tfb5 is required for Rpb9 mediated TC-NER. Taken together, our results indicate that Tfb5 is partially dispensable for Rad26 mediated TC-NER, especially in GG-NER deficient cells. However, this TFIIH subunit is required for other NER pathways.
PMCID: PMC2096704  PMID: 17644494
Rad7; Rad26; Rpb9; nucleotide excision repair; Saccharomyces cerevisiae; Tfb5; global genomic repair; transcription coupled repair
8.  A Role for CF1A 3′ End Processing Complex in Promoter-Associated Transcription 
PLoS Genetics  2013;9(8):e1003722.
The Cleavage Factor 1A (CF1A) complex, which is required for the termination of transcription in budding yeast, occupies the 3′ end of transcriptionally active genes. We recently demonstrated that CF1A subunits also crosslink to the 5′ end of genes during transcription. The presence of CF1A complex at the promoter suggested its possible involvement in the initiation/reinitiation of transcription. To check this possibility, we performed transcription run-on assay, RNAP II-density ChIP and strand-specific RT-PCR analysis in a mutant of CF1A subunit Clp1. As expected, RNAP II read through the termination signal in the temperature-sensitive mutant of clp1 at elevated temperature. The transcription readthrough phenotype was accompanied by a decrease in the density of RNAP II in the vicinity of the promoter region. With the exception of TFIIB and TFIIF, the recruitment of the general transcription factors onto the promoter, however, remained unaffected in the clp1 mutant. These results suggest that the CF1A complex affects the recruitment of RNAP II onto the promoter for reinitiation of transcription. Simultaneously, an increase in synthesis of promoter-initiated divergent antisense transcript was observed in the clp1 mutant, thereby implicating CF1A complex in providing directionality to the promoter-bound polymerase. Chromosome Conformation Capture (3C) analysis revealed a physical interaction of the promoter and terminator regions of a gene in the presence of a functional CF1A complex. Gene looping was completely abolished in the clp1 mutant. On the basis of these results, we propose that the CF1A-dependent recruitment of RNAP II onto the promoter for reinitiation and the regulation of directionality of promoter-associated transcription are accomplished through gene looping.
Author Summary
The termination of transcription requires two major multisubunit complexes in budding yeast. These termination complexes are localized at the 3′ end of genes. Recent studies have found the termination factors occupying the 5′ end of genes as well. In this study, we investigate the physiological role of a termination factor at the 5′ end of a gene. Our results show that the CF1 termination complex affects the recruitment of the transcription enzyme RNAP II onto the promoter for reinitiation of transcription. The complex also affects the directionality of transcription of the promoter-bound polymerase. We also found that the looped gene conformation was disrupted in the absence of a functional termination complex. The overall conclusion of these results is that the terminator-bound factors contact the 5′ end of genes due to gene looping, and affect both the recruitment of the polymerase at the promoter for reinitiation, and directionality of the promoter-initiated transcription. Thus, the role of termination factors is not restricted to the 3′ end of the gene, but they are also involved in promoter-associated transcription.
PMCID: PMC3744418  PMID: 23966880
9.  SCS3 and YFT2 Link Transcription of Phospholipid Biosynthetic Genes to ER Stress and the UPR 
PLoS Genetics  2012;8(8):e1002890.
The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER–resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.
Author Summary
The ability to form lipid droplets is a conserved property of eukaryotic cells that allows the storage of excess metabolic energy in a form that can be readily accessed. In adipose tissue, the storage of excess calories in lipid droplets normally protects other tissues from lipotoxicity and insulin resistance, but this protection is lost with chronic over-nutrition. The FAT storage-inducing transmembrane (FIT) proteins were recently identified as a conserved family of proteins that reside in the lipid bilayer of the endoplasmic reticulum and are implicated in lipid droplet formation. In this work we show that specific functions of the FIT proteins are conserved between yeast and humans and that SCS3 and YFT2, the yeast homologs of mammalian FIT2, are part of a large genetic interaction network connecting lipid metabolism, vesicle trafficking, transcription, and protein synthesis. From these interactions we determined that yeast strains lacking SCS3 and YFT2 are defective in their response to chronic ER stress and cannot induce the unfolded protein response pathway or transcription of phospholipid biosynthetic genes in low inositol. Our findings suggest that the mammalian FIT genes may play an important role in ER stress pathways, which are linked to obesity and type 2 diabetes.
PMCID: PMC3426550  PMID: 22927826
10.  Malleable Machines in Transcription Regulation: The Mediator Complex 
PLoS Computational Biology  2008;4(12):e1000243.
The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein–protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.
Author Summary
Intrinsically disordered proteins/regions do not adopt well-defined three dimensional structures; instead, they function as conformational ensembles. They are distinguished in molecular recognition and involved in various regulatory processes. Several components in the transcription machinery–for example, the transactivator domains of transcription factors–are disordered. Mediator, which is a large complex that transduces regulatory information from activators/repressors to the core apparatus, was found to contain a preponderance of intrinsically disordered regions in its various subunits. Such disordered regions are commonly involved in conformational changes coupled to functional transitions, in protein–protein interactions, or in posttranslational modifications. Several such predicted recognition sites were in good agreement with experimental data. Intrinsically disordered regions illuminate a novel aspect of Mediator's regulation and could explain its versatility and specificity in handling transcriptional signals. Their integral role in Mediator function is further underscored by the conserved arrangements of ordered/disordered segments and of the embedded interaction sites.
PMCID: PMC2588115  PMID: 19096501
11.  Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling 
Eukaryotic Cell  2004;3(2):264-276.
The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.
PMCID: PMC387650  PMID: 15075257
12.  Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis 
PLoS Pathogens  2012;8(11):e1002988.
The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.
Author Summary
Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia known as Legionnaires' disease. Following infection, the bacteria use a Type-IVB secretion system to translocate multiple effector proteins into macrophages and generate the Legionella-containing vacuole (LCV). The formation of the LCV involves the recruitment of specific bacterial effectors and host cell factors to the LCV as well as changes in its lipids composition. By screening L. pneumophila effectors for yeast growth inhibition, we have identified an effector, named LecE, that strongly inhibits yeast growth. By using yeast genetic tools, we found that LecE activates the yeast lipin homolog – Pah1, an enzyme that catalyzes the conversion of diacylglycerol to phosphatidic acid, these two molecules function as bioactive lipid signaling molecules in eukaryotic cells. In addition, by using yeast deletion mutants in genes relevant to lipids biosynthesis, we have identified another effector, named LpdA, which function as a phospholipase-D enzyme. Both effectors were found to be localized to the LCV during infection. Our results reveal a possible mechanism by which an intravacuolar pathogen might change the lipid composition of the vacuole in which it resides, a process that might lead to the recruitment of specific bacterial and host cell factors to the vacoule.
PMCID: PMC3486869  PMID: 23133385
13.  Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action 
Chemogenomic screens were performed in both budding and fission yeasts, allowing for a cross-species comparison of drug–gene interaction networks.Drug–module interactions were more conserved than individual drug–gene interactions.Combination of data from both species can improve drug–module predictions and helps identify a compound's mode of action.
Understanding the molecular effects of chemical compounds in living cells is an important step toward rational therapeutics. Drug discovery aims to find compounds that will target a specific pathway or pathogen with minimal side effects. However, even when an effective drug is found, its mode of action (MoA) is typically not well understood. The lack of knowledge regarding a drug's MoA makes the drug discovery process slow and rational therapeutics incredibly difficult. More recently, different high-throughput methods have been developed that attempt to discern how a compound exerts its effects in cells. One of these methods relies on measuring the growth of cells carrying different mutations in the presence of the compounds of interest, commonly referred to as chemogenomics (Wuster and Babu, 2008). The differential growth of the different mutants provides clues as to what the compounds target in the cell (Figure 2). For example, if a drug inhibits a branch in a vital two-branch pathway, then mutations in the second branch might result in cell death if the mutants are grown in the presence of the drug (Figure 2C). As these compound–mutant functional interactions are expected to be relatively rare, one can assume that the growth rate of a mutant–drug combination should generally be equal to the product of the growth rate of the untreated mutant with the growth rate of the drug-treated wild type. This expectation is defined as the neutral model and deviations from this provide a quantitative score that allow us to make informed predictions regarding a drug's MoA (Figure 2B; Parsons et al, 2006).
The availability of these high-throughput approaches now allows us to perform cross-species studies of functional interactions between compounds and genes. In this study, we have performed a quantitative analysis of compound–gene interactions for two fungal species (budding yeast (S. cerevisiae) and fission yeast (S. pombe)) that diverged from each other approximately 500–700 million years ago. A collection of 2957 compounds from the National Cancer Institute (NCI) were screened in both species for inhibition of wild-type cell growth. A total of 132 were found to be bioactive in both fungi and 9, along with 12 additional well-characterized drugs, were selected for subsequent screening. Mutant libraries of 727 and 438 gene deletions were used for S. cerevisiae and S. pombe, respectively, and these were selected based on availability of genetic interaction data from previous studies (Collins et al, 2007; Roguev et al, 2008; Fiedler et al, 2009) and contain an overlap of 190 one-to-one orthologs that can be directly compared. Deviations from the neutral expectation were quantified as drug–gene interactions scores (D-scores) for the 21 compounds against the deletion libraries. Replicates of both screens showed very high correlations (S. cerevisiae r=0.72, S. pombe r=0.76) and reproduced well previously known compound–gene interactions (Supplementary information). We then compared the D-scores for the 190 one-to-one orthologs present in the data set of both species. Despite the high reproducibility, we observed a very poor conservation of these compound–gene interaction scores across these species (r=0.13, Figure 4A).
Previous work had shown that, across these same species, genetic interactions within protein complexes were much more conserved than average genetic interactions (Roguev et al, 2008). Similarly we observed a higher cross-species conservation of the compound–module (complex or pathway) interactions than the overall compound–gene interactions. Specifically, the data derived from fission yeast were a poor predictor of S. cerevisaie drug–gene interactions, but a good predictor of budding yeast compound–module connections (Figure 4B). Also, a combined score from both species improved the prediction of compound–module interactions, above the accuracy observed with the S. cerevisae information alone, but this improvement was not observed for the prediction of drug–gene interactions (Figure 4B). Data from both species were used to predict drug–module interactions, and one specific interaction (compound NSC-207895 interaction with DNA repair complexes) was experimentally verified by showing that the compound activates the DNA damage repair pathway in three species (S. cerevisiae, S. pombe and H. sapiens).
To understand why the combination of chemogenomic data from two species might improve drug–module interaction predictions, we also analyzed previously published cross-species genetic–interaction data. We observed a significant correlation between the conservation of drug–gene and gene–gene interactions among the one-to-one orthologs (r=0.28, P-value=0.0078). Additionally, the strongest interactions of benomyl (a microtubule inhibitor) were to complexes that also had strong and conserved genetic interactions with microtubules (Figure 4C). We hypothesize that a significant number of the compound–gene interactions obtained from chemogenomic studies are not direct interactions with the physical target of the compounds, but include many indirect interactions that genetically interact with the main target(s). This would explain why the compound interaction networks show similar evolutionary patterns as the genetic interactions networks.
In summary, these results shed some light on the interplay between the evolution of genetic networks and the evolution of drug response. Understanding how genetic variability across different species might result in different sensitivity to drugs should improve our capacity to design treatments. Concretely, we hope that this line of research might one day help us create drugs and drug combinations that specifically affect a pathogen or diseased tissue, but not the host.
We present a cross-species chemogenomic screening platform using libraries of haploid deletion mutants from two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We screened a set of compounds of known and unknown mode of action (MoA) and derived quantitative drug scores (or D-scores), identifying mutants that are either sensitive or resistant to particular compounds. We found that compound–functional module relationships are more conserved than individual compound–gene interactions between these two species. Furthermore, we observed that combining data from both species allows for more accurate prediction of MoA. Finally, using this platform, we identified a novel small molecule that acts as a DNA damaging agent and demonstrate that its MoA is conserved in human cells.
PMCID: PMC3018166  PMID: 21179023
chemogenomics; evolution; modularity
14.  Nucleotide Excision Repair, Genome Stability, and Human Disease: New Insight from Model Systems 
Nucleotide excision repair (NER) is one of several DNA repair pathways that are universal throughout phylogeny. NER has a broad substrate specificity and is capable of removing several classes of lesions to the DNA, including those that accumulate upon exposure to UV radiation. The loss of this activity in NER-defective mutants gives rise to characteristic sensitivities to UV that, in humans, is manifested as a greatly elevated sensitivity to exposure to the sun. Xeroderma pigmentosum (XP), Cockaynes syndrome (CS), and trichothiodystrophy (TTD) are three, rare, recessively inherited human diseases that are linked to these defects. Interestingly, some of the symptoms in afflicted individuals appear to be due to defects in transcription, the result of the dual functionality of several components of the NER apparatus as parts of transcription factor IIH (TFIIH). Studies with several model systems have revealed that the genetic and biochemical features of NER are extraordinarily conserved in eukaryotes. One system that has been studied very closely is the budding yeast Saccharomyces cerevisiae. While many yeast NER mutants display the expected increases in UV sensitivity and defective transcription, other interesting phenotypes have also been observed. Elevated mutation and recombination rates, as well as increased frequencies of genome rearrangement by retrotransposon movement and recombination between short genomic sequences have been documented. The potential relevance of these novel phenotypes to disease in humans is discussed.
PMCID: PMC153785  PMID: 12488584
15.  Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein. 
Molecular and Cellular Biology  1995;15(6):3227-3237.
The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.
PMCID: PMC230555  PMID: 7760818
16.  Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex. 
Molecular and Cellular Biology  1995;15(7):3487-3495.
The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.
PMCID: PMC230585  PMID: 7791755
17.  Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III 
In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously.
Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related.
Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.
PMCID: PMC514540  PMID: 15298704
18.  A role for SUMO in nucleotide excision repair 
DNA repair  2011;10(12):1243-1251.
The two Siz/PIAS SUMO E3 ligases Siz1 and Siz2 are responsible for the vast majority of sumoylation in Saccharomyces cerevisiae. We found that siz1Δ siz2Δ mutants are sensitive to UV light. Epistasis analysis showed that the SIZ genes act in the nucleotide excision repair (NER) pathway, and suggested that they participate both in global genome repair (GGR) and in the Rpb9-dependent subpathway of transcription-coupled repair (TCR), but have minimal role in Rad26-dependent TCR. Quantitative analysis of NER at the single-nucleotide level showed that siz1Δ siz2Δ is deficient in repair of both the transcribed and non-transcribed strands of the DNA. These experiments confirmed that the SIZ genes participate in GGR. Their role in TCR remains unclear. It has been reported previously that mutants deficient for the SUMO conjugating enzyme Ubc9 contain reduced levels of Rad4, the yeast homolog of human XPC. However, our experiments do not support the conclusion that SUMO conjugation affects Rad4 levels. We found that several factors that participate in NER are sumoylated, including Rad4, Rad16, Rad7, Rad1, Rad10, Ssl2, Rad3, and Rpb4. Although Rad16 was heavily sumoylated, elimination of the major SUMO attachment sites in Rad16 had no detectable effect on UV resistance or removal of DNA lesions. SUMO attachment to most of these NER factors was significantly increased by DNA damage. Furthermore, SUMO-modified Rad4 accumulated in NER mutants that block the pathway downstream of Rad4, suggesting that SUMO becomes attached to Rad4 at a specific point during its functional cycle. Collectively, these results suggest that SIZ-dependent sumoylation may modulate the activity of multiple proteins to promote efficient NER.
PMCID: PMC3220943  PMID: 21968059
Smt3; ubiquitin; protein modification; protein degradation
19.  The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family. 
Nucleic Acids Research  1998;26(17):3908-3914.
Eukaryotic genes encoding ubiquitin-congugating enzyme (Ubc)-like proteins have been isolated from both human and yeast cells. The CROC-1 gene was isolated by its ability to transactivate c- fos expression in cell culture through a tandem repeat enhancer sequence. The yeast MMS2 gene was cloned by its ability to complement the methyl methanesulfonate sensitivity of the mms2-1 mutant and was later shown to be involved in DNA post-replication repair. We report here the identification of a human MMS2 ( hMMS2 ) cDNA encoding a novel human Ubc-like protein. hMMS2 and CROC-1 share >90% amino acid sequence identity, but their DNA probes hybridize to distinct transcripts. hMMS2 and CROC-1 also share approximately 50% identity and 75% similarity with the entire length of yeast Mms2. Unlike CROC-1 , whose transcript appears to be elevated in all tumor cell lines examined, the hMMS2 transcript is only elevated in some tumor cell lines. Collectively, these results indicate that eukaryotic cells may contain a highly conserved family of Ubc-like proteins that play roles in diverse cellular processes, ranging from DNA repair to signal transduction and cell differentiation. The hMMS2 and CROC-1 genes are able to functionally complement the yeast mms2 defects with regard to sensitivity to DNA damaging agents and spontaneous mutagenesis. Conversely, both MMS2 and hMMS2 were able to transactivate a c- fos - CAT reporter gene in Rat-1 cells in a transient co-transfection assay. We propose that either these proteins function in a common cellular process, such as DNA repair, or they exert their diverse biological roles through a similar biochemical interaction relative to ubiquitination.
PMCID: PMC147796  PMID: 9705497
20.  The yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription. 
Molecular and Cellular Biology  1995;15(4):2288-2293.
The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal transcription and NER. Our results also suggest that the repair and transcription functions of Tfb1 and Ssl1 are separable.
PMCID: PMC230456  PMID: 7891722
21.  HRAD1 and MRAD1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control gene. 
Nucleic Acids Research  1998;26(17):3971-3976.
Eukaryotic cells arrest at the G2checkpoint in the presence of DNA damage or incompletely replicated DNA. This cell cycle checkpoint prevents the development and propagation of genomic instability. In the fission yeast, this process requires the action of a number of genes, including rad1(+) . We report here the identification of human and mouse cDNAs that exhibit extensive sequence homology to rad1(+) . The human gene, called HRAD1 , encodes a 282 amino acid protein that is 27% identical and 53% similar to yeast Rad1p. The human homologue maintains its sequence similarity over the full length of the protein, including the three proposed 3'-->5' exonuclease domains, and the leucine rich repeat region. The mouse gene, called MRAD1 , encodes a 280 amino acid protein that is 90% identical and 96% similar to HRAD1 at the amino acid level. Expression of HRAD1 in yeast rad1 mutants partially restores radiation resistance and G2checkpoint proficiency to these mutants. Evolutionaryconservation of structure between HRAD1 , MRAD1 , rad1(+), Saccharomyces cerevisiae RAD17 and the Ustilago maydis REC1 checkpoint genes suggests that the function of the encoded proteins is conserved as well. The ability of HRAD1 to partially complement yeast rad1 mutants suggests that this gene is required for G2checkpoint control in human cells.
PMCID: PMC147814  PMID: 9705507
22.  A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase 
BMC Systems Biology  2010;4:125.
Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary.
Orthologous gene pairs were ranked by order statistics according to their co-expression profiles across species, allowing us to infer conserved meiotic genes despite obvious differences in cellular synchronicity and composition in organisms. We demonstrated that conserved co-expression networks could successfully recover known meiotic genes, including homologous recombination genes, chromatin cohesion genes, and genes regulating meiotic entry. We also showed that conserved co-expression pairs exhibit functional connections, as evidenced by the annotation similarity in Gene Ontology and overlap with physical interactions. More importantly, we predicted six new meiotic genes through their co-expression linkages with known meiotic genes, and subsequently used the genetically more amenable yeast system for experimental validation. The deletion mutants of all six genes showed sporulation defects, equivalent to a 100% validation rate.
We identified evolutionarily conserved gene modules in meiotic prophase by integrating cross-species and cross-sex expression profiles from budding yeast, mouse, and human. Our co-expression linkage analyses confirmed known meiotic genes and identified several novel genes that might be critical players in meiosis in multiple species. These results demonstrate that our approach is highly efficient to discover evolutionarily conserved novel meiotic genes, and yeast can serve as a valuable model system for investigating mammalian meiotic prophase.
PMCID: PMC2944139  PMID: 20819218
23.  RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery 
PLoS Genetics  2013;9(6):e1003543.
Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development.
Author Summary
It is well known that splicing occurs cotranscriptionally but the functional coupling between splicing and transcription has not been studied carefully in the context of a multicellular organism in development. We took advantage of the amenable C. elegans genetics and genomics to demonstrate a functional relationship between RSR-2, whose yeast and human orthologs are components of the spliceosome, and transcription. Although we found that RSR-2 interacts with proteins present in the spliceosome, moderate inhibition of rsr-2 by RNAi did not significantly affect splicing, but rather caused a decrease in transcript levels that was critical for germline sex determination. Our investigation on such a paradox of a spliceosomal component affecting transcription resulted in several lines of evidence linking RSR-2 with transcription: (i) RSR-2 immunoprecipitates chromatin resembling the ChIP-Seq profile of RNAPII, (ii) RSR-2 is present in intronless genes, (iii) rsr-2(RNAi) globally modifies the distribution of RNAPII along genes and its phosphorylation state, (iv) RSR-2 coimmunoprecipitates with RNAPII, and (v) RSR-2 interacts with PRP-19, which is a component of the spliceosome required for efficient transcriptional activity. Our findings raise an intriguing question: to what extent does a moderate alteration in some spliceosome components affect the gene expression process by perturbing splicing or transcription?
PMCID: PMC3675011  PMID: 23754964
24.  Mutation of RNA Pol III Subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and Disrupts Zebrafish Digestive Development  
PLoS Biology  2007;5(11):e312.
The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-Δ yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function.
Author Summary
The transmission of genetic information from DNA to messenger RNA to protein depends on the function of a large number of small noncoding RNA molecules. The genes encoding these RNAs are transcribed by RNA polymerase III (Pol III), a 17-subunit protein complex whose structure is closely related to that of RNA polymerases I and II. Here, we report the effect of a mutation in a gene encoding one Pol III subunit, Polr3b, which disrupts proliferation and growth of tissue progenitor cells in the zebrafish digestive system. Analyses of a nearly identical mutation in the yeast S. pombe gene encoding Polr3b, also known as Rpc2, suggested that the zebrafish mutation disrupted the mutant Polr3b protein's interaction with another Pol III subunit, Polr3k, also known as Rpc11. Overexpression of the gene encoding Polr3k in the Polr3b mutants partially rescued (reversed) the mutant phenotype. These findings extend our knowledge of the mechanism of Pol III function, which appears to have been highly conserved during eukaryotic evolution. Furthermore, these data also suggest that assembly of the 17-subunit Pol III enzyme is a dynamic process, since Polr3k overexpression can partially rescue the mutant phenotype. Understanding how Pol III is assembled has implications for human disease, since Pol III activity is markedly increased in most cancers.
Transcription of tRNAs and other noncoding RNAs by RNA polymerase III is essential for cell proliferation, growth, and survival. With the aide of experiments in the fission yeast,S. pombe, the authors report how a small in-frame deletion in the second largest Pol III subunit, Polr3b, affects tissue progenitor cells in the developing zebrafish digestive system.
PMCID: PMC2229849  PMID: 18044988
25.  Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts 
Nucleic Acids Research  2001;29(14):3123-3130.
Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) β, ζ or η. Base excision repair was also proficient without Polβ. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polα (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Polδ (pol3-1) or Polɛ (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Polδ and Polɛ, respectively. Deleting the polymerase catalytic domain of Polɛ (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Polɛ, but not by purified yeast Polη. These results suggest that efficient repair synthesis of yeast NER requires both Polδ and Polɛ in vitro, and that the low fidelity Polη is not accessible to repair synthesis during NER.
PMCID: PMC55800  PMID: 11452038

Results 1-25 (1310098)