Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

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Homologous and heterologous regulation of somatostatin-binding sites on chicken adenohypophysial membranes

Journal of Endocrinology ◽

10.1677/joe.0.1270417 ◽

1990 ◽

Vol 127 (3) ◽

pp. 417-425 ◽

Cited By ~ 4

Author(s):

S. Harvey ◽

J. S. Baidwan ◽

D. Attardo

Keyword(s):

Pituitary Gland ◽

Binding Sites ◽

Recombinant Dna ◽

Inhibitory Effect ◽

Partial Recovery ◽

Caudal Lobe ◽

Direct Inhibitory Effect ◽

Pituitary Glands

ABSTRACT Binding of 125I-labelled [Tyr1]-somatostatin (125I-[Tyr1]-SRIF) to pituitary caudal lobe membranes was suppressed in immature chickens 1 and 2 h after i.v. administration of unlabelled SRIF at concentrations of 1–100 μg/kg. In-vitro preincubation of chicken pituitary glands for 0·5–4·0 h with 0·1 μmol SRIF/l similarly reduced the binding of 125I-[Tyr1]-SRIF to caudal lobe membrane preparations. After a 4-h incubation in 0·1 mmol SRIF/l, the withdrawal of SRIF from the incubation media was accompanied 4 h later by a partial recovery in the binding of 125I-[Tyr1]-SRIF to pituitary membranes. Passive immunoneutralization of endogenous SRIF resulted in a prompt (within 1 h) and sustained (for at least 24 h) suppression of 125I-[Tyr1]-SRIF binding to pituitary membranes. The i.m. administration of cysteamine (300 mg/kg) to 12-week-old birds depleted hypothalamic SRIF stores and decreased the density of 125I-[Tyr1]-SRIF-binding sites in the caudal and cephalic lobes of the chicken pituitary gland. The reduction in SRIF content and in SRIF-binding sites occurred within 1 h of cysteamine administration and was maintained for at least 24 h. In 6-week-old birds, cysteamine (300 mg/kg) administration suppressed pituitary binding of 125I-[Tyr1]-SRIF for at least 5 days. Circulati concentrations of GH were markedly decreased 1 and 4 h after cysteamine injection, but not after 24 h. Pituitary binding sites for 125I-[Tyr1]-SRIF were not affected by pretreatment of pituitary glands for 2–12 h in vitro with thyroxine or oestradiol-17β (1 nmol/l–10 μmol/l) or with ovine GH or recombinant DNA-derived chicken GH (1–100 μg/ml in vitro and 100–1000 μg/kg in vivo). Ovine prolactin, at concentrations of 1–100 μg/ml was also without effect on 125I-[Tyr1]-SRIF binding to pituitary membranes following a 2- or 4-h incubation with pituitary glands. Pituitary binding sites for 125I-[Tyr1]-SRIF were, however, increased after a 24-h incubation with 1 μmol tri-iodothyronine (T3)/l in vitro and 4 and 24 h after the administration of T3 (100–1000 μg/kg) in vivo. Although T3 had no direct inhibitory effect on 125I-[Tyr1]-SRIF binding to pituitary membranes, binding was suppressed 1 and 2 h after the in-vivo administration of T3 at concentrations of 100–1000 μg/kg. These results therefore demonstrate homologous and heterologous regulation of SRIF-binding sites in the chicken pituitary gland. Journal of Endocrinology (1990) 127, 417–425

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Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.5134 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5134-5142 ◽

Cited By ~ 393

Author(s):

M Kozak

Keyword(s):

Secondary Structure ◽

Ribosomal Subunit ◽

Loop Structure ◽

Entry Site ◽

Range Interaction ◽

Stem Loop ◽

Stem Loop Structure ◽

Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

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TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

10.1101/480525 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kyster K. Nanan ◽

David M. Sturgill ◽

Maria F. Prigge ◽

Morgan Thenoz ◽

Allissa A. Dillman ◽

...

Keyword(s):

Binding Sites ◽

Genomic Dna ◽

Multiple Roles ◽

Ctcf Binding ◽

Cellular Model ◽

Potential Mechanism ◽

Dynamic Regulation ◽

Genome Wide

SummaryThe mechanisms supporting dynamic regulation of CTCF binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC) as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified ∼13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

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Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

10.1101/2021.04.12.439580 ◽

2021 ◽

Author(s):

Fei Liu ◽

Amy C.H Lee ◽

Fang Guo ◽

Andrew S. Kondratowicz ◽

Holly M Micolochick Steuer ◽

...

Keyword(s):

Dominant Role ◽

Regulatory Element ◽

Rna Stability ◽

Antiviral Effect ◽

Inhibitory Effect ◽

Stem Loop ◽

Knock Out ◽

The Individual

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize HBV RNA via the interaction with the viral post-transcriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, HBsAg production and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies, therefore it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (EC50 ranged from 1.4 to 6.8 nM) and in vivo by 0.93 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity towards the inhibitory effect of AB-452. Although neither single knock-out (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO could not result in any measurable phenotypic changes, but displays a similar antiviral effect as AB-452 treatment when PAPD5 is depleted simultaneously. PAPD5/7 double KO confers complete resistance to AB-452 treatment. Our results thus indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5 targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication.

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Possible Mode of Action of Ticlopidine: A Novel Inhibitor of Platelet Aggregation

10.1055/s-0039-1687403 ◽

1979 ◽

Cited By ~ 1

Author(s):

H. Johnson ◽

J. B. Heywood

Keyword(s):

Platelet Aggregation ◽

Mode Of Action ◽

Potent Inhibitor ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Thromboxane Synthetase ◽

Sustained Effect ◽

Dependent Inhibition

Ticlopidine (T) is weakly active in vitro, but is a potent inhibitor of platelet aggregation induced by ADP, collagen, thrombin, adrenaline, arachidonic acid, prostaglandin (PG) endoperoxide and thromboxane A2 with a sustained effect, when administered to a variety of animal species, including man. Platelets from treated animals were normal in ultrastructure and 14C-ADP binding was not modified by T. Basal PG synthesis was unaffected, whereas aspirin (A) had a marked inhibitory effect. Platelet cyclo-oxygenase and thromboxane synthetase activities were 90.6±12.9 and 96.1±5.3% of control following T treatment. In contrast to A, T had no effect on vascularprostacyclin (PGI2) synthesis, this being 1.4±0.1, 0.5±0.1 and 1.3±0. 3ng/mg wet weight aorta in T and A-treated and control animals respectively. Platelets from T-treated rats were significantly more responsive to inhibition by exogenous PGI2 (0.2-4 ng/ml) and PGE1 (4- 20 ng/ml). when compared with controls. T administration (30-300 mg/kg) resulted in a dose-dependent inhibition of ADP-induced platelet aggregation (26.0- 87. 5%) and enhancement of platelet reactivity to PGI2 (37.0-159.8%). There was a good correlation between these parameters (r=+0.994). T is a potent inhibitor of platelet aggregati on with a novel mode of action. It is not aspirin-like, but may act to potentiate endogenous PGI2 in vivo, possibly through an effect on platelet adenylate cyclase.

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Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.5134-5142.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5134-5142

Author(s):

M Kozak

Keyword(s):

Secondary Structure ◽

Ribosomal Subunit ◽

Loop Structure ◽

Entry Site ◽

Range Interaction ◽

Stem Loop ◽

Stem Loop Structure ◽

Delta G

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Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.835 ◽

1999 ◽

Vol 19 (1) ◽

pp. 835-845 ◽

Cited By ~ 52

Author(s):

Zeng-Feng Wang ◽

Thomas C. Ingledue ◽

Zbigniew Dominski ◽

Ricardo Sanchez ◽

William F. Marzluff

Keyword(s):

Translational Control ◽

Mrna Processing ◽

Loop Structure ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Stem Loop Structure ◽

Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

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A novel plant-derived inhibitor of cAMP-mediated fluid and chloride secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.1.g58 ◽

1999 ◽

Vol 276 (1) ◽

pp. G58-G63 ◽

Cited By ~ 16

Author(s):

S. E. Gabriel ◽

S. E. Davenport ◽

R. J. Steagall ◽

V. Vimal ◽

T. Carlson ◽

...

Keyword(s):

Cholera Toxin ◽

Ussing Chamber ◽

Fluid Secretion ◽

Chloride Secretion ◽

Inhibitory Effect ◽

Adult Mice ◽

Dependent Inhibition ◽

Chamber Studies

We have identified an agent (SP-303) that shows efficacy against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl−secretion. Administration of cholera toxin to adult mice results in an increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals, respectively). This elevation in FA induced by cholera toxin was significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment. Moreover, when SP-303 was administered 3 h after cholera toxin, a dose-dependent inhibition of FA levels was observed with a half-maximal inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or T84 monolayer preparations, SP-303 had a significant effect on both basal current and forskolin-stimulated Cl−current. SP-303 also induced an increase in resistance that paralleled the observed decrease in current. These data suggest that SP-303 has an inhibitory effect on cAMP-mediated Cl−and fluid secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.

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Lu 23051, A New Potent Inhibitor Of Platelet Aggregation

10.1055/s-0038-1653265 ◽

1981 ◽

Author(s):

H D Lehmann ◽

J Gries ◽

D Lenke

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Coronary Vessels ◽

Inhibitory Effect ◽

Spontaneously Hypertensive ◽

Dependent Inhibition ◽

Induced Aggregation

6- [p-(2-(Chiorpropionylamino)phenyl] -4.5-dihydro-5-methyl-3(2H)-pyridazinone, LU 23051, is primarily characterized by its strong inhibition of platelet aggregation under in vitro and in vivo conditions. In vitro there is a concentration-dependent inhibition of ADP and collagen induced aggregation in platelet rich plasma of man, rat and dog. The inhibitory concentration EC 33 % is 0.0010-0.030 mg/1 (man: ADP-0.030, col 1.-0.013 mg/l) depending on species and type of aggregation. When administered orally in ex vivo experiments on rats and dogs the substance is found to have a dose-dependent antiaggregatory effect in the range from 0.1-3.16 mg/kg. The ED 33 % is 0.27-0.63 mg/kg.-In addition after oral administration the substance has a good inhibitory effect in models being based on intravascular platelet aggregation. Thus, a dose of 1 mg/kg inhibits laser-induced aggregation in mesenteric venules of rats. Mortality after i.v. injection of collagen in mice is reduced by 50 % after a dose of 0.02 mg/kg. A dose of 0.039 mg/kg prolongs the bleeding time of rats by 50 %. The aggregation-inhibiting action is of long duration (0.1 mg/kg p.o.∼24 h). The substance does not interfere with clotting.Besides its effect on platelet aggregation LU 23051 acts as vasodilatator as well. Dilatation of coronary vessels by 100 % is seen in isolated guinea-pig hearts at a concentration of 0.1 mg/l. In spontaneously hypertensive rats the substance has an anti hypertensive effect. The ED 20 % is 0.36 mg/kg p.o.The combination of antiaggregatory and vasodilatatory effects opens up interesting aspects with respect to the pharmacotherapeutic use of the new substance

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An intranasal ASO therapeutic targeting SARS-CoV-2

10.1101/2021.05.17.444397 ◽

2021 ◽

Author(s):

Chi Zhu ◽

Justin Y. Lee ◽

Jia Z. Woo ◽

Lei Xu ◽

Xammy Nguyenla ◽

...

Keyword(s):

Viral Replication ◽

Locked Nucleic Acid ◽

Rna Sequences ◽

Stem Loop ◽

Stem Loop Structure ◽

Promising Therapeutic Approach ◽

Vaccine Distribution ◽

Rapid Deployment

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 "variants of concern" tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.

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