A Method for Isolation of Intact, Translationally Active Ribonucleic Acid

Cathala, Guy; Savouret, J.-F.; Méndez, Bernardita; West, Brian L.; Karin, Michael; Martial, Joseph; Baxter, John D.

doi:10.1089/dna.1983.2.329

Cited by 1,461 publications

(547 citation statements)

References 16 publications

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“…IF2mt1, IF2mt2 and control cells were plated at 2.0 Â 10 5 cells/ ml, treated for 24 or 48 h by human IFNa2 (500 U/ml) or IFNa2 and chloramphenicol (50 mg/ml) and collected for RNA extraction using the guanidine thiocyanate-lithium chloride procedure. 37 Total RNAs (20 mg) were analyzed by Northern blot. 36 After transfer onto nylon membranes (Appligen, Illkirch, Graffenstaden, France), rRNA were revealed with 0.5 M Na acetate (pH 5), 0.04% (w/v) methylene blue.…”

Section: Methodsmentioning

confidence: 99%

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

et al. 2007

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Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2 0 -5 0 oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2 0 -5 0 oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNa-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNa-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNa and partially reverses IFNa-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt. Apoptosis is a regulated cell death process that plays a central role in the control of many physiological events. Cells die by apoptosis during embryonic development, tissue homeostasis or immune regulation and defects in the apoptotic pathway during these events can lead to excessive cell accumulation with dramatic consequences. 1-3 Interferons (IFNs) are among the regulators of apoptosis. They belong to a family of cytokines produced and secreted by mammalian cells in response to various inducers. They are negative regulators of cell proliferation through induction of cell-cycle arrest and apoptosis, but the mechanisms are not yet fully understood. The 2-5A/RNase L pathway is a single-stranded RNA (ssRNA) decay pathway induced by IFNs: the 2-5A synthetases are induced by IFNs and upon activation by doublestranded RNA (dsRNA), convert ATP into a series of oligomers known as 2 0 -5 0 oligoadenylates (2-5A). 4,5 The 2-5A activates RNase L, a latent endoribonuclease, which inhibits protein synthesis by cleaving ssRNA. 6,7 RNase L plays a central role in IFNs cell growth inhibition and in IFN-induced apoptosis. [8][9][10][11][12] Activation of RNase L causes caspase-dependent apoptosis accompanied by cytochrome c release from the mitochondria. 13 20 RNase L activity is regulated not only by 2-5A binding, but also by interaction with other proteins. It has been shown previously that RNase L can form a heterodimer with the RNase L inhibitor (RLI), a protein that inhibits 2-5A binding to RNase L, resulting in an inhibition of RNase L activity. 21,22 More recently, we demonstrated that RNase L interacts with the translation termination release factor eRF3/GSPT1 and that their interaction is important for its role in translation termination regulation. 23 We set out to identify other potential RNase L partners to better comprehend its mechanism of action. In the present study, a yeast two-hybrid screening, using human RNase L as bait identifies ...

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Section: Methodsmentioning

confidence: 99%

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

et al. 2007

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“…R N A was isolated with guanidine thiocyanate as RNase inhibitor by the procedure of Cathala [5] with minor modifications. All plant tissues including the microspores were frozen in liquid nitrogen and ground in a prechilled mortar.…”

Section: Nucleic Acid Isolationmentioning

confidence: 99%

Isolation and characterization of a microspore-specific gene from tobacco

et al. 1996

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The characterization of a gene with a unique microspore-specific expression pattern is reported. Isolated microspores from tobacco were used to synthesize a cDNA library. Clones that did not hybridize to leaf cDNA were further characterized by northern analysis. One clone proved to be a microspore-specific cDNA, representing a transcript of 650 nt. The corresponding gene, NTM19 (Nicotiana tabacum microspore-specific), was isolated and its sequence analysed. The gene encodes a protein of 10.8 kDa with a pI of 6.92 and a putative signal sequence at the N-terminus. A localization study revealed a unique spatial and temporal distribution. The transcript was only detected in the unicellular microspore. No hybridization signals were observed in other pollen developmental stages, nor in the surrounding anther tissues or other vegetative tissues of the plant. Therefore it can be concluded that NTM 19 is a gene with a highly microspore-specific character according to both localization and stage of expression. Southern blot analysis demonstrated the presence of a small gene family. The occurrence of TNM 19 was investigated in a range of closely and distantly related species and was found to be present in other solanaceous species, including the ancestors of tobacco and in a monocot species.

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“…precipitation Total RNA was isolated using the guanidine monothiocyanate extraction procedure of Cathala et al [13]. Poly(A+) RNA was prepared with poly(U) Sepharose according to [14].…”

Section: Isolation Of Poiy(a ') Rna In Vitro Translation and Immuno-mentioning

confidence: 99%

Retinoic acid controls expression of epidermal transglutaminase at the pre‐translational level

Michel¹,

Reichert²,

Isnard³

et al. 1989

FEBS Letters

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Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca*+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.Cl revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.

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A Method for Isolation of Intact, Translationally Active Ribonucleic Acid

Cited by 1,461 publications

References 16 publications

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

Isolation and characterization of a microspore-specific gene from tobacco

Retinoic acid controls expression of epidermal transglutaminase at the pre‐translational level

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