scholarly journals RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP)

2020 ◽  
Author(s):  
Laura Moreno-Leon ◽  
Emma L West ◽  
Michelle O’Hara-Wright ◽  
Linjing Li ◽  
Rohini Nair ◽  
...  
Keyword(s):  
Steady State ◽  
Retinitis Pigmentosa ◽  
Relative Importance ◽  
Wild Type ◽  
Disease Pathogenesis ◽  
Therapeutic Outcomes ◽  
Alternatively Spliced ◽  
Steady State Levels ◽  
Retinitis Pigmentosa Gtpase Regulator ◽  
Normal Controls

Abstract Mutations in RPGR (retinitis pigmentosa GTPase regulator) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15 are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 mRNAs are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals Quinolone Resistance Due to Reduced Target Enzyme Expression

2003 ◽  
Vol 185 (23) ◽  
pp. 6883-6892 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Steady State ◽  
Quinolone Resistance ◽  
Start Codon ◽  
Cell Multiplication ◽  
Compensatory Mechanism ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Resistance Phenotype ◽  
Allelic Exchange ◽  
Steady State Levels

ABSTRACT We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G→A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41°C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41°C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

scholarly journals Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA.

1996 ◽  
Vol 16 (8) ◽  
pp. 4426-4435 ◽  
Author(s):  
O Kessler ◽  
L A Chasin
Keyword(s):  
Steady State ◽  
Actinomycin D ◽  
Chinese Hamster ◽  
Nuclear Pore ◽  
Mrna Levels ◽  
Wild Type ◽  
Adenine Phosphoribosyltransferase ◽  
Aprt Gene ◽  
Steady State Levels ◽  
Nonsense Codons

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in four of the five exons of the adenine phosphoribosyltransferase (aprt) gene and have found a pattern of mRNA reduction similar to that seen in systems studied previously: a decrease in steady-state mRNA levels of 5- to 10-fold for mutations in exons 1, 2, and 4 but little effect for mutations in the 3'-most exon (exon 5). Nuclear aprt mRNA levels showed a similar decrease. Nonsense-containing aprt mRNA decayed at the same rate as wild-type mRNA in these cell lines after inhibition of transcription with actinomycin D. Nonsense-containing aprt mRNA is associated with polysomes, ruling out a model in which stable residual mRNA escapes degradation by avoiding translation initiation. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNAs without globally inhibiting transcription. In contrast to measurements made in the presence of actinomycin D, after inhibition of aprt transcription with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic aprt mRNA seen here could account for the nonsense-mediated reduction in steady-state levels of aprt mRNA. However, the low levels of nonsense-bearing aprt mRNA in the nucleus suggest a sensibility of mRNA to translation or translatability before it exits that compartment. Quantitation of the steady-state levels of transcripts containing introns revealed no accumulation of partially spliced aprt RNA and hence no indication of nonsense-mediated aberrancies in splicing. Our results are consistent with a model in which translation facilitates the export of mRNA through a nuclear pore. However, the mechanism of this intriguing nucleocytoplasmic communication remains to be determined.

Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells

1986 ◽  
Vol 6 (8) ◽  
pp. 2865-2871
Author(s):  
L McConlogue ◽  
S L Dana ◽  
P Coffino
Keyword(s):  
Steady State ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Mrna Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Cells Of Origin ◽  
Multistep Process ◽  
Steady State Levels ◽  
Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

2003 ◽  
Vol 285 (2) ◽  
pp. H866-H874 ◽  
Author(s):  
Craig R. Hampton ◽  
Akira Shimamoto ◽  
Christine L. Rothnie ◽  
Jeaneatte Griscavage-Ennis ◽  
Albert Chong ◽  
...  
Keyword(s):  
Steady State ◽  
Infarct Size ◽  
Ischemic Preconditioning ◽  
Tissue Injury ◽  
Late Phase ◽  
Wild Type ◽  
Hsp70 Protein ◽  
Steady State Levels ◽  
Shock Proteins

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(–/–)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(–/–) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(–/–) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.

scholarly journals Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (11) ◽  
pp. 3954-3964 ◽  
Author(s):  
F S Genbauffe ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Normal Control ◽  
Catabolite Repression ◽  
Gene Products ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Recombinant Plasmids ◽  
Control Pattern ◽  
Steady State Levels

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3954-3964
Author(s):  
F S Genbauffe ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Normal Control ◽  
Catabolite Repression ◽  
Gene Products ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Recombinant Plasmids ◽  
Control Pattern ◽  
Steady State Levels

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

scholarly journals Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

1986 ◽  
Vol 6 (8) ◽  
pp. 2865-2871 ◽  
Author(s):  
L McConlogue ◽  
S L Dana ◽  
P Coffino
Keyword(s):  
Steady State ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Mrna Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Cells Of Origin ◽  
Multistep Process ◽  
Steady State Levels ◽  
Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

scholarly journals Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

1998 ◽  
Vol 72 (8) ◽  
pp. 6325-6331 ◽  
Author(s):  
Baoling Ying ◽  
Kimberley Smith ◽  
Katherine R. Spindler
Keyword(s):  
Steady State ◽  
Adenovirus Type ◽  
Wild Type Virus ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Wild Type ◽  
Cultured Fibroblasts ◽  
Early Region ◽  
Steady State Levels

ABSTRACT Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+,Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 andpmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.

scholarly journals Argonaute binding within 3′-untranslated regions poorly predicts gene repression

2020 ◽  
Author(s):  
Yongjun Chu ◽  
Audrius Kilikevicius ◽  
Jing Liu ◽  
Krystal C Johnson ◽  
Shinnichi Yokota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Mammalian Cells ◽  
Gene Repression ◽  
Type Versus ◽  
Untranslated Regions ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Knock Out ◽  
Steady State Levels

Abstract Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3′-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3′-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

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