Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

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Study of Staphylococcus aureusPathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

Infection and Immunity ◽

10.1128/iai.69.2.657-664.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 657-664 ◽

Cited By ~ 32

Author(s):

P. Stutzmann Meier ◽

J. M. Entenza ◽

P. Vaudaux ◽

P. Francioli ◽

M. P. Glauser ◽

...

Keyword(s):

Single Gene ◽

Individual Factors ◽

Gene Products ◽

Streptococcus Gordonii ◽

Pathogenic Role ◽

Gain Of Function ◽

Function Strategy

ABSTRACT Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordoniiwas more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deletingclfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity ofclfA-positive streptococci when both clfA andcoa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

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Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5315 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5315-5323 ◽

Cited By ~ 37

Author(s):

J Imbert ◽

M Zafarullah ◽

V C Culotta ◽

L Gedamu ◽

D Hamer

Keyword(s):

Gene Transcription ◽

Regulatory Elements ◽

Gene Promoters ◽

Direct Role ◽

Transcription In Vitro ◽

Mouse Cells ◽

Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

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Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

10.1101/2020.12.29.424694 ◽

2020 ◽

Author(s):

Sophie H. L. Austin ◽

Lachlan Harris ◽

Oana Paun ◽

Piero Rigo ◽

François Guillemot ◽

...

Keyword(s):

Stem Cell ◽

Beta Catenin ◽

Dependent Manner ◽

Direct Role ◽

Adult Nscs ◽

Hippocampal Networks ◽

Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

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FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.625715 ◽

2021 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Marianna Kapetanou ◽

Tobias Nespital ◽

Luke S. Tain ◽

Andre Pahl ◽

Linda Partridge ◽

...

Keyword(s):

Proteasome Activity ◽

20S Proteasome ◽

Direct Role ◽

Forkhead Box ◽

Age Related ◽

Proteasome Subunits ◽

Rate Limiting

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

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Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5315-5323.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5315-5323

Author(s):

J Imbert ◽

M Zafarullah ◽

V C Culotta ◽

L Gedamu ◽

D Hamer

Keyword(s):

Gene Transcription ◽

Regulatory Elements ◽

Gene Promoters ◽

Direct Role ◽

Transcription In Vitro ◽

Mouse Cells ◽

Affinity Reagent

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The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.2.253 ◽

2000 ◽

Vol 191 (2) ◽

pp. 253-264 ◽

Cited By ~ 226

Author(s):

Jos Domen ◽

Samuel H. Cheshier ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Cells ◽

Wild Type ◽

Direct Role ◽

Hematopoietic Stem

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2–transgenic mice have increased numbers of HSC in bone marrow (2.4× wild type), but fewer of these cells are in the S/G2/M phases of the cell cycle (0.6× wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

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RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2379 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2379-2393 ◽

Cited By ~ 46

Author(s):

T H Chang ◽

M W Clark ◽

A J Lustig ◽

M E Cusick ◽

J Abelson

Keyword(s):

Cell Nucleus ◽

Specific Antibody ◽

Essential Role ◽

Mrna Splicing ◽

Temperature Sensitive ◽

Gene Products ◽

Nonpermissive Temperature ◽

Exogenous Rna

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.

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PRP18, a protein required for the second reaction in pre-mRNA splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.324 ◽

1990 ◽

Vol 10 (1) ◽

pp. 324-332 ◽

Cited By ~ 36

Author(s):

U Vijayraghavan ◽

J Abelson

Keyword(s):

Micrococcal Nuclease ◽

Heat Inactivation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Wild Type ◽

Exon 1 ◽

In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

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Alpha Enolase 1 Ubiquitination and Degradation Mediated by Ehrlichia chaffeensis TRP120 Disrupts Glycolytic Flux and Promotes Infection

Pathogens ◽

10.3390/pathogens10080962 ◽

2021 ◽

Vol 10 (8) ◽

pp. 962

Author(s):

Bing Zhu ◽

Jere W. McBride

Keyword(s):

Host Cell ◽

Ectopic Expression ◽

Metabolic Reprogramming ◽

Glycolytic Flux ◽

Ehrlichia Chaffeensis ◽

Direct Role ◽

Cell Processes

Ehrlichia chaffeensis modulates numerous host cell processes, including gene transcription to promote infection of the mononuclear phagocyte. Modulation of these host cell processes is directed through E. chaffeensis effectors, including TRP120. We previously reported that TRP120 moonlights as a HECT E3 Ub ligase that ubiquitinates host cell transcription and fate regulators (PCGF5 and FBW7) to promote infection. In this study, we identified a novel TRP120 substrate and examined the relationship between TRP120 and α-enolase (ENO1), a metalloenzyme that catalyzes glycolytic pathway substrate dehydration. Immunofluorescence microscopy and coimmunoprecipitation demonstrated interaction between ENO1 and TRP120, and ubiquitination of ENO-1 by TRP120 was detected in vivo and in vitro. Further, ENO-1 degradation was observed during infection and was inhibited by the proteasomal inhibitor bortezomib. A direct role of TRP120 Ub ligase activity in ENO-1 degradation was demonstrated and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. siRNA knockdown of ENO-1 coincided with increased E. chaffeensis infection and ENO-1 knockdown disrupted glycolytic flux by decreasing the levels of pyruvate and lactate that may contribute to changes in host cell metabolism that promote infection. In addition, we elucidated a functional role of TRP120 auto-ubiquitination as an activating event that facilitates the recruitment of the UbcH5 E2 ubiquitin-conjugating enzyme. This investigation further expands the repertoire of TRP120 substrates and extends the potential role of TRP120 Ub ligase in infection to include metabolic reprogramming.

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Requirement for the Budding Yeast Polo Kinase Cdc5 in Proper Microtubule Growth and Dynamics

Eukaryotic Cell ◽

10.1128/ec.00283-07 ◽

2008 ◽

Vol 7 (3) ◽

pp. 444-453 ◽

Cited By ~ 24

Author(s):

Chong J. Park ◽

Jung-Eun Park ◽

Tatiana S. Karpova ◽

Nak-Kyun Soung ◽

Li-Rong Yu ◽

...

Keyword(s):

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Growth Defect ◽

Temperature Sensitive ◽

Independent Manner ◽

Polo Kinase ◽

Early Anaphase

ABSTRACT In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.

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