RESUMO
Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.
Assuntos
Íntrons , Fenótipo , Splicing de RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Spliceossomos , Spliceossomos/metabolismo , Spliceossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Histonas/metabolismo , Histonas/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0137820.].
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Some bee species use wax to build their nests. They store honey and raise their brood in cells made entirely from wax. How can the bee brood breathe and develop properly when sealed in wax cells? We compared the chemical composition and structural properties of the honey cappings and worker brood cappings of the honeybee Apis mellifera carnica, measured the worker brood respiration, and calculated the CO2 gradients across the two types of cappings. We identified microscopic pores present in the brood cappings that allow efficient gas exchange of the developing brood. In contrary, honey cappings are nearly gas impermeable to protect honey from fermenting. Similar principles apply in bumble bees. Our data suggest the control of gas exchange of cappings as a selective pressure in the evolution of wax-building bees that drives their adaptation for using wax in two highly contrasting biological contexts.
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Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs. Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role. The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.
Assuntos
Genes Fúngicos , Íntrons , Kluyveromyces/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.
Assuntos
Splicing de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Íntrons , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.
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Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Mitose/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Biotina/metabolismo , DNA Fúngico/metabolismo , Epistasia Genética , Mutação/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcrição GênicaRESUMO
BACKGROUND: Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. CONCLUSIONS/SIGNIFICANCE: Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast.
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Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocinese , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética , Mitose , Proteínas de Schizosaccharomyces pombe/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family.
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Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Ciclo Celular , Sequência Conservada , DNA Fúngico/metabolismo , Genes Reporter/genética , Mutação , Elementos de Resposta/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5' and 3' splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.
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Processamento Alternativo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Spliceossomos/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Luz , Mutação , Fotoperíodo , Filogenia , Desenvolvimento Vegetal , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Repressoras , Spliceossomos/genética , Temperatura , Fatores de Transcrição/genéticaRESUMO
Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.
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Íntrons , Sítios de Splice de RNA , Splicing de RNA , RNA Fúngico/química , Saccharomyces cerevisiae/genética , Ascomicetos/genética , Sequência de Bases , Cofilina 1/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
BACKGROUND: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. CONCLUSIONS/SIGNIFICANCE: This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.
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Motivos de Aminoácidos , Proteínas Fúngicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Sequência Conservada/genética , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Fosforilação , Filogenia , Reação em Cadeia da Polimerase , Ligação Proteica , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have found that a high iron concentration in solid complete cultivation medium potentiates cell-cell and cell-surface adhesion of the fission yeast Schizosaccharomyces pombe. Spotted giant colonies grown on iron-rich media were found to be more compact and more resistant to washing than those grown on plates with a standard iron content. Furthermore, we have documented that excess environmental iron stimulates the invasive growth of S. pombe (and Saccharomyces cerevisiae). Three-dimensional, branched, washing-resistant structures composed mostly of elongated, but separate fission yeast cells, were formed within the solid agar medium. The degree of both adhesion and invasion displayed a specific, iron concentration-dependent response. Our results suggest a novel link between iron availability and the intensively studied and important fungal virulence factors, adhesion and invasion.
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Adesão Celular/fisiologia , Meios de Cultura/química , Hifas/crescimento & desenvolvimento , Ferro/farmacologia , Schizosaccharomyces/crescimento & desenvolvimento , Ágar , Ferro/metabolismo , Técnicas Microbiológicas , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologiaRESUMO
Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2-associated spliceosomal complexes purified from prp45(1-169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1-169) cells revealed that branch point mutants accumulated more pre-mRNA whereas 5' and 3' splice site mutants showed elevated levels of lariat-exon intermediate as compared to wild-type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119-379) in prp45(1-169) cells restored Prp22 partition in the Cwc2-pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1-169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step-proofreading helicase Prp22, to splicing efficiency of substrates non-conforming to the consensus.
Assuntos
RNA Helicases DEAD-box/metabolismo , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Alelos , Sequência de Aminoácidos , RNA Helicases DEAD-box/genética , Íntrons , Dados de Sequência Molecular , Mutação , Fenótipo , Precursores de RNA/metabolismo , Fatores de Processamento de RNA , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11+ and cbf12+, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11+ and cbf12+ are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTGA/GGAA in vitro. The deletion of cbf11+ is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.
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Divisão do Núcleo Celular/fisiologia , Modelos Biológicos , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Adesão Celular , Divisão do Núcleo Celular/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de SinaisRESUMO
BACKGROUND: The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. RESULTS: We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. CONCLUSION: Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.
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Proteínas Fúngicas/genética , Fungos/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Evolução Molecular , Proteínas Fúngicas/química , Fungos/química , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Schizosaccharomyces/química , Schizosaccharomyces/genética , Alinhamento de Sequência , Fatores de Transcrição/químicaAssuntos
Sequência Rica em At , Artefatos , Cromatografia em Agarose/métodos , DNA/química , DNA/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Sequências Repetitivas de Ácido Nucleico , Soluções Tampão , DNA/genética , Mutação , Desnaturação de Ácido Nucleico , Controle de QualidadeRESUMO
The essential gene product Prp45 (379 aa) of Saccharomyces cerevisiae is a highly conserved, but N-terminally abridged, ortholog of the human transcriptional coactivator SKIP, which is involved in TGFbeta, Notch, and steroid hormone signaling. We used a diploid strain harboring PRP45 deletion, which is inviable in the haploid, to test for complementation with the truncated versions of Prp45. The N-terminal half of the protein (aa 1 to 190), denoted as the SNW domain, was found sufficient to support the essential function. Interestingly, substituting the SNW motif itself with AAA was compatible with viability. GFP-tagged Prp45 was localized in nuclear "speckles" over a diffuse nuclear background. We further found that Prp45 activated the transcription of a reporter gene in S. cerevisiae when targeted to DNA. The observed effect relied in part upon the presence of conserved helical repeats and upon the highly charged C-terminal domain (pI = 11.3). Prp45, which lacks most of the binding motifs of the human ortholog, and whose N-terminal half is sufficient for supporting the growth of prp45 cells, might be helpful in elucidating the essential function of SNW/SKIP proteins.
Assuntos
Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência Conservada , Regulação Fúngica da Expressão Gênica , Genes Reporter , Teste de Complementação Genética , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Mapeamento de Peptídeos , Plasmídeos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Transfecção/métodosRESUMO
The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae. Swapping the regulatory domains between SRA and PRC revealed that N117D, I96T, and V124L mutations in the n-src- and RT-loops of the SH3 domain of PRC are responsible for the low kinase activity of PRC. Moreover, introducing D117N, R95W, T96I, and L124V into activated c-Src(Y527F) caused a 2.5-fold increase in its activity. The mutations in the CD linker KP249,250DG and L255A, which were shown to activate c-Src, had no effect on the activity of the "SH2-activated" Src kinases. Together our data suggest that in the "SH2-activated" forms of Src, the SH3 domain continues to influence the kinase activity via the direct contacts of the n-src- and RT-loops with the kinase N-terminal lobe.