Korneeva, E.I.1, Lagarkova, M.A.2,3, Kiselev, S.L.3 and Korneev, S.A.1
1 - Sussex Centre for Neuroscience, University of Sussex, Brighton, UK. 2 - Institute of Gene Biology, Moscow, Russia. 3 - Vavilov Institute of General Genetics, Moscow, Russia; e-mail: s.korneev@sussex.ac.uk)
Until recently, non-coding natural antisense transcripts (NATs) in eukaryotic systems were thought to be a minor group of unimportant transcripts. However, recent experiments have identified a surprisingly large number of NATs in a variety of organisms including mammals. Moreover, a role in the regulation of gene expression has been reported for a number of these NATs /for review, see 1 and 2/.
Our previous work on a molluscan model system Lymnaea stagnalis has shown that a NAT complementary to the nitric oxide synthase (NOS)-encoding mRNA plays an important role in the regulation of the nitric oxide (NO) signalling pathway /3, 4/. This NAT is transcribed from a pseudogene evolved from a duplicated copy of NOS gene by internal DNA inversion /5/.
We have now extended our studies to mammalian systems in the hope of revealing conserved mechanisms regulating the production of NO in humans. In mammals NO is produced by a family of NOSs, which is composed of three major isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS) and the macrophage inducible NOS (iNOS). Importantly, we have discovered a locus in the human genome whose structure has been affected by an internal DNA inversion reminiscent of our finding in Lymnaea. The locus is located on the chromosome 17 and has the highest similarity to the iNOS gene. Hereafter we will refer to this locus as anti-iNOS. The chain of events that led to the creation of the NOS pseudogene in molluscs and the anti-iNOS locus in humans is identical - gene duplication was followed by an internal DNA inversion. This remarkable evolutionary conservatism suggests strongly that the human anti-iNOS locus, like its molluscan equivalent, could also produce antisense RNA molecules with potential role in the regulation of NO production. In order to test the hypothesis we used recently isolated undifferentiated human embryonic stem cell (hESC) lines /6/.
Importantly, RT-PCR analysis performed on these cells has confirmed our suggestion. The anti-iNOS locus is indeed transcribed in hESCs as a NAT containing a region of significant complementarity to iNOS mRNA. Moreover, we have also found that the levels of anti-iNOS and iNOS gene expression in hESCs lines are comparable. Taking into account the proposed role of endogenous NO in the modulation of neural cell differentiation, we have extended our studies and measured, by means of quantitative real time RT-PCR, the expression of the anti-iNOS NAT in undifferentiated stem cells and in cells, which were induced to differentiate into neurogenic precursors such as neurospheres. Two established cell lines ESM01 and ESM02 were used for this analysis. These lines maintained a normal karyotype, were viable after freezing and thawing and they exhibited no significant differences in morphology, proliferation rate or stability during more than 50 passages. Neurospheres were generated from hESCs lines using previously published protocols. Remarkably, the results of our quantitative experiments were very similar for both lines and demonstrated an approximately 6-fold decrease in the expression of the anti-iNOS NAT in neurospheres in comparison to undifferentiated cells. These data are in line with previous observations that showed differentiating and antiproliferating activity of endogenous NO in many cell types including embryonic and postnatal neural precursor cells /for review, see 7/. They also suggest that the anti-iNOS NAT can be involved in the differentiation of hESCs through the negative regulation of NOS gene expression.
1. Cao, X., Yeo, G., Muotri, A.R., Kuwabara, T. and Gage, F.H. (2006) Noncoding RNAs in the mammalian central nervous system. Annu. Rev. Neurosci., 29: 77-103.
2. Korneev, S. and O’Shea, M. (2005) Natural antisense RNAs in the nervous system. Rev. Neurosci., 16: 213-222.
3. Korneev, S.A., Park, J.H. and O'Shea, M. (1999) Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by an antisense RNA transcribed from an NOS pseudogene. J. Neurosci., 19: 7711-7720.
4. Korneev, S.A., Straub, V., Kemenes, I., Korneeva, E.I., Ott, S.R., Benjamin, P.R. and O'Shea, M. (2005) Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation. J. Neurosci., 25: 1188-1192.
5. Korneev, S. and O’Shea, M. (2002) Evolution of nitric oxide synthase regulatory genes by DNA inversion. Mol. Biol. Evol., 19: 1228-1233.
6. Lagarkova, M.A., Volchkov, P.Y., Lyakisheva, A.V., Philonenko, E.S., Kiselev, S.L. (2006) Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle, 5: 416-420.
7. Estrada, C. and Murillo-Carretero, M. (2005) Nitric oxide and adult neurogenesis in health and disease. The Neuroscientist, 11: 294-307.
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