scholarly journals Impaired Ribosome Biogenesis Disrupts the Integration between Morphogenesis and Nuclear Duplication during the Germination of Aspergillus fumigatus

2008 ◽  
Vol 7 (4) ◽  
pp. 575-583 ◽  
Author(s):  
Ruchi Bhabhra ◽  
Daryl L. Richie ◽  
H. Stanley Kim ◽  
William C. Nierman ◽  
Jarrod Fortwendel ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Compensatory Response ◽  
Translational Machinery ◽  
Germination Process ◽  
Polarized Cell Growth ◽  
Polysome Profile ◽  
Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37°C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a ΔcgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the ΔcgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although ΔcgrA conidia germinated normally at 22°C, they swelled excessively when incubated at 37°C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.

scholarly journals Sfp1 Plays a Key Role in Yeast Ribosome Biogenesis

2003 ◽  
Vol 2 (5) ◽  
pp. 1061-1068 ◽  
Author(s):  
Ian Fingerman ◽  
Vijayalakshmi Nagaraj ◽  
David Norris ◽  
Andrew K. Vershon
Keyword(s):  
Ribosome Biogenesis ◽  
Zinc Finger Protein ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Reporter Construct ◽  
Promoter Elements ◽  
Translational Machinery ◽  
Finger Protein ◽  
Activation Of Transcription ◽  
Direct Binding

ABSTRACT Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect. sfp1 cells have a reduced size and a slow growth phenotype. In this study we show that SFP1 plays a role in ribosome biogenesis. An sfp1 strain is hypersensitive to drugs that inhibit translational machinery. sfp1 strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed SFP1 induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is SFP1 dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.

scholarly journals Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

2016 ◽  
Vol 8 (6) ◽  
pp. 579-588 ◽  
Author(s):  
Ninette Genster ◽  
Elisabeth Præstekjær Cramer ◽  
Anne Rosbjerg ◽  
Katrine Pilely ◽  
Jack Bernard Cowland ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Host Defense ◽  
Knockout Mice ◽  
Fungal Infections ◽  
Inflammatory Cells ◽  
Lectin Pathway ◽  
Wild Type ◽  
Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

scholarly journals Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

2021 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic Gillet ◽  
Kerstin Doerner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic C Gillet ◽  
Kerstin Dörner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals Deletion of the Regulatory Subunit of Protein Kinase A in Aspergillus fumigatus Alters Morphology, Sensitivity to Oxidative Damage, andVirulence

2006 ◽  
Vol 74 (8) ◽  
pp. 4865-4874 ◽  
Author(s):  
Wei Zhao ◽  
John C. Panepinto ◽  
Jarrod R. Fortwendel ◽  
Lauren Fox ◽  
Brian G. Oliver ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Oxidative Damage ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complemented mutant. Both growth and germination rates of the mutant are reduced, and there are morphological abnormalities in conidiophores, leading to reduced conidiation. Conidia from the ΔpkaR mutant are more sensitive to killing by hydrogen peroxide, menadione, paraquat, and diamide. However, the hyphae of the mutant are killed to a greater extent only by paraquat and diamide, whereas they are less susceptible to the effects of hydrogen peroxide. In an immunosuppressed mouse model, intranasally administered conidia of the mutant are significantly less virulent than those of the wild type or a complemented mutant. Unregulated PKA signaling is detrimental to the virulence of A. fumigatus, perhaps through the reduced susceptibility of the mutant to damage by oxidizing agents and reduced growth kinetics.

scholarly journals The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

2021 ◽  
Author(s):  
Marianthi Kiparaki ◽  
Chaitali Khan ◽  
Virginia Folgado Marco ◽  
Jacky Chuen ◽  
Nicholas E Baker
Keyword(s):  
Transcription Factor ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Cell Competition ◽  
Translation Rate ◽  
Eif2α Phosphorylation ◽  
Bzip Domain ◽  
Domain Protein ◽  
Global Translation

Ribosomal Protein (Rp) gene haploinsufficiency affects overall translation rate, leads to cell elimination by competition with wild type cells in mosaic tissues, and sometimes leads to accumulation of protein aggregates. The changes in ribosomal subunit levels observed are not sufficient for these effects, which all depend on the AT-hook, bZip domain protein Xrp1. In Rp+/- cells, Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α. eIF2α phosphorylation was sufficient to reduce translation in, and also enable cell competition of, otherwise wild type cells. Unexpectedly, however, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF1α1, or eIF5A), also increased eIF2α phosphorylation and enabled cell competition. In all cases this was through the Xrp1 expression that was induced, placing Xrp1 as the downstream instigator of cell competition that also contributed to overall translation deficits. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Thus, Xrp1, which is shown here to be a sequence-specific transcription factor, is the master regulator that triggers cell competition and other consequences of multiple ribosomal stresses.

scholarly journals Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

2012 ◽  
Vol 11 (11) ◽  
pp. 1333-1344 ◽  
Author(s):  
Isabelle Raymond-Bouchard ◽  
Cassandra S. Carroll ◽  
Jason R. Nesbitt ◽  
Kevin A. Henry ◽  
Linda J. Pinto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Mutant Proteins ◽  
Opportunistic Fungal Pathogen ◽  
Triacetylfusarinine C ◽  
Siderophore Transporter

ABSTRACTSiderophores have been identified as virulence factors in the opportunistic fungal pathogenAspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophoreN,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids ofA. fumigatusMirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in theSaccharomyces cerevisiaestrain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using55Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization inA. fumigatusshowed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.

scholarly journals Ribosomal Protein L33 Is Required for Ribosome Biogenesis, Subunit Joining, and Repression of GCN4 Translation

2007 ◽  
Vol 27 (17) ◽  
pp. 5968-5985 ◽  
Author(s):  
Pilar Martín-Marcos ◽  
Alan G. Hinnebusch ◽  
Mercedes Tamame
Keyword(s):  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Upstream Open Reading Frame ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
Reading Frame ◽  
Efficient Processing ◽  
Subunit Joining ◽  
General Translation

ABSTRACT We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd− phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37°C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (ΔA). At 28°C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in ΔA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by ΔA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAi Met suppresses the Gcd− phenotype of rpl33a-G76R cells, dissociation of tRNAi Met from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.

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