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A novel long non-coding natural antisense RNA is a negative regulator of Nos1 gene expression.

Korneev SA, Maconochie M, Naskar S, Korneeva EI, Richardson GP, O'Shea M - Sci Rep (2015)

Bottom Line: Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown.Nos1 produces nitric oxide (NO), a major signaling molecule in the CNS implicated in many important functions including neuronal differentiation and memory formation.We show that the newly discovered NAT negatively regulates Nos1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Sussex Neuroscience, School of Life Science, University of Sussex, Brighton BN1 9QG, UK.

ABSTRACT
Long non-coding natural antisense transcripts (NATs) are widespread in eukaryotic species. Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown. Here we report that a long NAT (Mm-antiNos1 RNA) complementary to mRNA encoding the neuronal isoform of nitric oxide synthase (Nos1) is expressed in the mouse brain and is transcribed from the non-template strand of the Nos1 locus. Nos1 produces nitric oxide (NO), a major signaling molecule in the CNS implicated in many important functions including neuronal differentiation and memory formation. We show that the newly discovered NAT negatively regulates Nos1 gene expression. Moreover, our quantitative studies of the temporal expression profiles of Mm-antiNos1 RNA in the mouse brain during embryonic development and postnatal life indicate that it may be involved in the regulation of NO-dependent neurogenesis.

No MeSH data available.


Mm-antiNos1 RNA down-regulates Nos1 protein synthesis.(a) Representative Western blot analysis of cultured Neuro2a cells incubated with transfection mixtures containing either 0.2 μg (lane 1) or 1 μg (lane 2) of pcDNA 3.1/Mm-antiNos1. Non-transfected control cells are shown in lane C. (b) Quantitative densitometric analysis of the Western blot shown in a. Values for the Nos1 expression were quantified relative to the expression of β-actin. Finally, the values for Nos1 expression in transfected Neuro2a cells were normalised to Nos1 protein levels in non-transfected cells.
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f3: Mm-antiNos1 RNA down-regulates Nos1 protein synthesis.(a) Representative Western blot analysis of cultured Neuro2a cells incubated with transfection mixtures containing either 0.2 μg (lane 1) or 1 μg (lane 2) of pcDNA 3.1/Mm-antiNos1. Non-transfected control cells are shown in lane C. (b) Quantitative densitometric analysis of the Western blot shown in a. Values for the Nos1 expression were quantified relative to the expression of β-actin. Finally, the values for Nos1 expression in transfected Neuro2a cells were normalised to Nos1 protein levels in non-transfected cells.

Mentions: To examine the impact of Mm-antiNos1 RNA on Nos1 gene expression we overexpressed this NAT in Neuro2a cells. First we engineered an expression construct containing the full length Mm-antiNos1 cDNA. We then transiently transfected Neuro2a cells with this Mm-antiNos1 expression construct using Lipofectamine 2000. Success of the transfection experiments has been confirmed by the results of real-time RT-PCR showing significant up-regulation of Mm-antiNos1 RNA in the transfected cells (data not shown). It is of importance that the quantitative analysis did not detect any changes in the expression level of Nos1 mRNA in cells overexpressing Mm-antiNos1 RNA (data not shown). Remarkably, however, our experiments showed that the overexpression of Mm-antiNos1 RNA caused a substantial reduction of Nos1 protein produced (Fig. 3). Thus we can conclude that Mm-antiNos1 RNA negatively regulates Nos1 gene expression and does so post-transcriptionally.


A novel long non-coding natural antisense RNA is a negative regulator of Nos1 gene expression.

Korneev SA, Maconochie M, Naskar S, Korneeva EI, Richardson GP, O'Shea M - Sci Rep (2015)

Mm-antiNos1 RNA down-regulates Nos1 protein synthesis.(a) Representative Western blot analysis of cultured Neuro2a cells incubated with transfection mixtures containing either 0.2 μg (lane 1) or 1 μg (lane 2) of pcDNA 3.1/Mm-antiNos1. Non-transfected control cells are shown in lane C. (b) Quantitative densitometric analysis of the Western blot shown in a. Values for the Nos1 expression were quantified relative to the expression of β-actin. Finally, the values for Nos1 expression in transfected Neuro2a cells were normalised to Nos1 protein levels in non-transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495418&req=5

f3: Mm-antiNos1 RNA down-regulates Nos1 protein synthesis.(a) Representative Western blot analysis of cultured Neuro2a cells incubated with transfection mixtures containing either 0.2 μg (lane 1) or 1 μg (lane 2) of pcDNA 3.1/Mm-antiNos1. Non-transfected control cells are shown in lane C. (b) Quantitative densitometric analysis of the Western blot shown in a. Values for the Nos1 expression were quantified relative to the expression of β-actin. Finally, the values for Nos1 expression in transfected Neuro2a cells were normalised to Nos1 protein levels in non-transfected cells.
Mentions: To examine the impact of Mm-antiNos1 RNA on Nos1 gene expression we overexpressed this NAT in Neuro2a cells. First we engineered an expression construct containing the full length Mm-antiNos1 cDNA. We then transiently transfected Neuro2a cells with this Mm-antiNos1 expression construct using Lipofectamine 2000. Success of the transfection experiments has been confirmed by the results of real-time RT-PCR showing significant up-regulation of Mm-antiNos1 RNA in the transfected cells (data not shown). It is of importance that the quantitative analysis did not detect any changes in the expression level of Nos1 mRNA in cells overexpressing Mm-antiNos1 RNA (data not shown). Remarkably, however, our experiments showed that the overexpression of Mm-antiNos1 RNA caused a substantial reduction of Nos1 protein produced (Fig. 3). Thus we can conclude that Mm-antiNos1 RNA negatively regulates Nos1 gene expression and does so post-transcriptionally.

Bottom Line: Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown.Nos1 produces nitric oxide (NO), a major signaling molecule in the CNS implicated in many important functions including neuronal differentiation and memory formation.We show that the newly discovered NAT negatively regulates Nos1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Sussex Neuroscience, School of Life Science, University of Sussex, Brighton BN1 9QG, UK.

ABSTRACT
Long non-coding natural antisense transcripts (NATs) are widespread in eukaryotic species. Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown. Here we report that a long NAT (Mm-antiNos1 RNA) complementary to mRNA encoding the neuronal isoform of nitric oxide synthase (Nos1) is expressed in the mouse brain and is transcribed from the non-template strand of the Nos1 locus. Nos1 produces nitric oxide (NO), a major signaling molecule in the CNS implicated in many important functions including neuronal differentiation and memory formation. We show that the newly discovered NAT negatively regulates Nos1 gene expression. Moreover, our quantitative studies of the temporal expression profiles of Mm-antiNos1 RNA in the mouse brain during embryonic development and postnatal life indicate that it may be involved in the regulation of NO-dependent neurogenesis.

No MeSH data available.