Cyclosporin A-resistance based gene placement system for Neurospora crassa

Bardiya, Nirmala; Shiu, Patrick

doi:10.1016/j.fgb.2006.12.011

Cited by 77 publications

(85 citation statements)

References 31 publications

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“…These observations suggest that a concentration-dependent specific inhibition of PaCYPD, but not of other cyclophilins, via the interaction with CSA is responsible for the observed effect on growth. Moreover, in accordance with earlier findings reported for N. crassa (Tropschug et al, 1989;Bardiya & Shiu, 2007), the PaCYPD ⁄ CSA interaction product appears to inhibit the growth of P. anserina cultures. Therefore, at high CSA concentrations, the high abundance of PaCYPD in overexpressing strains results in a much stronger growth inhibition than in the wild-type strain.…”

Section: Effects Of Cyclosporin a Treatmentsupporting

confidence: 92%

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

et al. 2010

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SummaryCyclophilin D (CYPD) is a mitochondrial peptidyl prolyl-cis,trans-isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wildtype with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.

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Section: Effects Of Cyclosporin a Treatmentsupporting

confidence: 92%

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

et al. 2010

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“…Because y14 (NCU03226, Supercontig 1: 4971616-4972891 +) was closely linked to his-3 (NCU03139, Supercontig 1, 4694797-4697762 +), for analyses of reporter activity in Dy14, a two-step approach was used for placing the reporter at a different chromosomal position in this strain. First, luciferase constructs were placed at csr-1 (NCU00726, Supercontig 1, 7404112-7406331 2) by transformation of the wild-type strain (FGSC 2489) with luc reporters on linearized plasmids designed for targeting to csr-1 (Bardiya and Shiu 2007). Disruption of this locus results in resistance to cyclosporin A.…”

Section: Strainsmentioning

confidence: 99%

“…For integrating the luc reporters at csr-1, plasmids pZY122 (csr-1::cox-5 P \cox-5\luc\cox-5), pZY123 (csr-1::cox-5 P \cox-5\ luc\eIF4A3+I), and pZY124 (csr-1::cox-5 P \cox-5\luc\eIF4A3-I) were created by placing the NotI-ClaI restriction fragments of pYZ82, pZY92, and pZY94, respectively, that contained the reporter genes into the corresponding sites of plasmid pCSR-1 (Bardiya and Shiu 2007).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3′UTR Introns

Zhang¹,

Sachs

2015

Genetics

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In higher eukaryotes the accelerated degradation of mRNAs harboring premature termination codons is controlled by nonsense-mediated mRNA decay (NMD), exon junction complex (EJC), and nuclear cap-binding complex (CBC) factors, but the mechanistic basis for this quality-control system and the specific roles of the individual factors remain unclear. Using Neurospora crassa as a model system, we analyzed the mechanisms by which NMD is induced by spliced 39-UTR introns or upstream open reading frames and observed that the former requires NMD, EJC, and CBC factors whereas the latter requires only the NMD factors. The transcripts for EJC components eIF4A3 and Y14, and translation termination factor eRF1, contain spliced 39-UTR introns and each was stabilized in NMD, EJC, and CBC mutants. Reporter mRNAs containing spliced 39-UTR introns, but not matched intronless controls, were stabilized in these mutants and were enriched in mRNPs immunopurified from wild-type cells with antibody directed against human Y14, demonstrating a direct role for spliced 39-UTR introns in triggering EJC-mediated NMD. These results demonstrate conclusively that NMD, EJC, and CBC factors have essential roles in controlling mRNA stability and that, based on differential requirements for these factors, there are branched mechanisms for NMD. They demonstrate for the first time autoregulatory control of expression at the level of mRNA stability through the EJC/CBC branch of NMD for EJC core components, eIF4A3 and Y14, and for eRF1, which recognizes termination codons. Finally, these results show that EJC-mediated NMD occurs in fungi and thus is an evolutionarily conserved quality-control mechanism.KEYWORDS nonsense-mediated mRNA decay (NMD); RNA stability; exon junction complex (EJC); cap-binding complex (CBC); spliced 39-UTR intron; post-transcriptional control; ribosome; translation; Neurospora crassa T HE nonsense-mediated mRNA decay (NMD) pathway targets mRNA for degradation through the recognition of translated termination codons determined to be premature (Nicholson et al. 2009;Kervestin and Jacobson 2012;Popp and Maquat 2013). The NMD pathway can degrade mRNAs that contain upstream open reading frames (uORFs) or long 39-UTRs. The termination-related processes that trigger uORFmediated or long 39-UTR-mediated NMD are considered to be similar (Amrani et al. 2004(Amrani et al. , 2006Nicholson et al. 2009) in that termination events occurring far from the context created by the mRNA poly(A) tail can result in mRNA degradation (the "faux-UTR" model). In higher eukaryotes, NMD can also act on mRNAs that contain an intron downstream of a termination codon, such as aberrantly spliced transcripts or 39-UTR introncontaining transcripts (Sauliere et al. 2010;Bicknell et al. 2012). When a spliced intron is positioned at least 50-55 nt downstream of a termination codon, the exon junction complex (EJC) positioned near the exon-exon junction is not displaced by translating ribosomes and its continued association with mRNA targets the mRNA for deg...

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“…To facilitate creation of green fluorescent protein (GFP) gene fusion strains for colocalization and complementation studies, the pCSR1 vector (35), which allows positive selection of csr-1 targeted transformants, was modified to contain the Myceliophthora thermophila gpdA promoter, a multiple cloning site (MCS), the synthetic GFP (sGFP) gene, and the M. thermophila gpdA terminator (T. Starr and N. L. Glass, personal communication).…”

Section: Methodsmentioning

confidence: 99%

“…Transformation of N. crassa by electroporation was performed as previously described (35). The sGFP gene fusions were transformed into the respective gene deletion strains.…”

Section: Methodsmentioning

confidence: 99%

Physiological Role of Acyl Coenzyme A Synthetase Homologs in Lipid Metabolism in Neurospora crassa

et al. 2013

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Acyl coenzyme A (CoA) synthetase (ACS) enzymes catalyze the activation of free fatty acids (FAs) to CoA esters by a two-step thioesterification reaction. Activated FAs participate in a variety of anabolic and catabolic lipid metabolic pathways, including de novo complex lipid biosynthesis, FA ␤-oxidation, and lipid membrane remodeling. Analysis of the genome sequence of the filamentous fungus Neurospora crassa identified seven putative fatty ACSs (ACS-1 through ACS-7). ACS-3 was found to be the major activator for exogenous FAs for anabolic lipid metabolic pathways, and consistent with this finding, ACS-3 localized to the endoplasmic reticulum, plasma membrane, and septa. Double-mutant analyses confirmed partial functional redundancy of ACS-2 and ACS-3. ACS-5 was determined to function in siderophore biosynthesis, indicating alternative functions for ACS enzymes in addition to fatty acid metabolism. The N. crassa ACSs involved in activation of FAs for catabolism were not specifically defined, presumably due to functional redundancy of several of ACSs for catabolism of exogenous FAs. Lipids are essential components of all living cells. They are major components of biological membranes (e.g., phospholipids) and serve as energy reserves (e.g., triacylglycerols) as well as signaling molecules (e.g., sphingolipids and lysophosphatidic acid). In fungi, fatty acids (FAs) are synthesized de novo via the FA synthase complex (FAS type I) present in the cytosol (1) or in the mitochondria via a suite of enzymes, including an acyl carrier protein. These mitochondrial FAS type II enzymes are not present in a complex (2). Many fungi can also utilize exogenous FAs, both for incorporation into lipids and as a carbon source. In addition to incorporation into complex lipids in the endoplasmic reticulum (ER) (3, 4) and mitochondria (5), FAs can be remodeled by desaturation and elongation (6-8) and degraded for energy production in the peroxisome (glyoxysome) (9, 10) and in the mitochondria (9, 11).One of the most important catalytic activities in lipid metabolism is activation of FAs by acyl coenzyme A (CoA) synthetase (ACS) (EC 6.2.1.3). Activated FAs participate in a number of cellular metabolic pathways, including synthesis of phospholipids, triacylglycerols, and cholesterol esters, FA elongation, FA desaturation, and ␤-oxidation. ACSs catalyze the two-step ATP-dependent reaction of FA activation, whereby first the FA substrate is adenylated to form an acyl-AMP intermediate and subsequently AMP is exchanged with CoA, forming a thioester bond to yield the activated acyl-CoA (12).The number of carbons present in the FA substrate of ACSs ranges from 2 to more than 26. The characteristic length of the FA substrate defines subfamilies of ACSs as short-chain (SC; 2 to 4 carbons), medium-chain (MC; 4 to 12 carbons), long-chain (LC; 12 to 20 carbons), very-long-chain (VLC; 18 to 26 carbons), and "bubblegum" (14 to 24 carbons) FA activators (13). All ACSs contain a highly conserved ATP/AMP binding motif (14), which is conserved among all...

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Cyclosporin A-resistance based gene placement system for Neurospora crassa

Cited by 77 publications

References 31 publications

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3′UTR Introns

Physiological Role of Acyl Coenzyme A Synthetase Homologs in Lipid Metabolism in Neurospora crassa

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