Transcript Identification a Novel Regulatory Mechanism Governing One of the

Identification a Novel Regulatory Mechanism Governing One of the Key Stem Cell
Pluripotency Genes, SOX2, Mediated by the Non-coding RNA SOX2OT
Alireza Shahryari1,2, Mahshid Malakootian2, Mohammad Shafiee3, Mohammad Vasei4, Seyed Javad Mowla1,2*
1ParsGenome
Company, Tehran, Iran; 2Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares
University, Tehran, Iran; 3Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology,
Tehran, Iran; 4Department of Pathology, Faculty of Medicine, Shariati Hospital, Tehran University of Medical Sciences, Tehran,
Iran
Abstract
The SOX2 gene, a master regulator of pluripotency, is embedded within the
third intron of a long non-coding RNA (lncRNA) known as SOX2 overlapping
transcript (SOX2OT). Here, we showed the lncRNA SOX2OT is implicated in
positive regulation of SOX2 expression in NT2 stem cells. We also identified
seven novel splice variants of SOX2OT that expressed in stem cells and
some human cancers such as brain cancers. All together, our data provide
a novel regulatory mechanism governing one of key stem cell pluripotency
genes, SOX2, mediated by the lncRNA SOX2OT.
Materials and methods
Two pairs of siRNAs targeting human SOX2OT were employed to suppress
SOX2OT expression in NT2 cells. NT2 cells were treated with all-trans
retinoic acid (ATRA) to induce its differentiation into the neuron-like cells,
and gene expressions and cell cycle alterations were examined. To block
RNA polymerase, NT2 cells were also treated with actinomycin D.
Expression relative to
scrambled siRNA
0.3
0.25
SOX2OT
0.2
SOX2
0.15
0.1
0.05
0
siRNA1 siRNA2
% of cells in each phase
Results and discussion
Suppression of SOX2OT caused a significant declination of SOX2
expression (0.1, P< 0.01) and also induced a G1 cell cycle arrest in NT2 cells
(Fig.2 ). Subcellular localization study showed that SOX2OT is a nuclear
RNA, and it differentially spliced and produced two novel variants (SOX2OTS1 and SOX2OT-S2) which co-expressed with SOX2 in NT2 cells.
90
80
scrambled siRNA
70
SOX2OT siRNA
60
50
40
Moreover, SOX2OT-S2 was found to be the most stable transcript among
SOX2OT transcripts. We then evaluated all ESTs of SOX2OT depositing in
GenBank implying existing of several potential splice variants for SOX2OT.
Using different sets of primers we found novel splice variants of lncRNA
SOX2DOT (SOX2DOT-S1, SOX2DOT-S2, SOX2DOT-S3, SOX2DOT-S4 and
SOX2DOT-S5) (Fig.3). Expression study of SOX2OT and SOX2DOT following
the process of neural differentiation of NT2 stem cells by ATRA revealed a
decrease pattern of SOX2OT and an increase expression level of SOX2DOT
during four weeks of differentiation, suggesting their different potential
functional links to the undifferentiated and differentiated state of the NT2
cells respectively (Fig.4).
Expression relative to
GAPDH
Figure1: The Intron-less gene of SOX2 holds in the 3rd intron of lncRNA
SOX2OT
Figure3: Schematic representative of SOX2OT gene and novel variants of
SOX2DOT
Expression relative to
GAPDH
Introduction
Long noncoding RNAs (lncRNAs) are a newly discovered class of ncRNAs,
some of them regulate stem cell pluripotency and neurogenesis. Recent
studies revealed that the expression of several lncRNAs correlates with the
expression of pluripotency regulators such as OCT4 and NANOG (1).
Interestingly, the SOX2 gene lies within an intron of the lncRNA SOX2OT
(Fig.1) (2). SOX2DOT (SOX2 distal overlapping transcript) is an isoform of
SOX2OT transcribed from a region located >550 kb upstream of SOX2 is
expressed in brain (3). In this study, we investigated a potential function of
SOX2OT in human embryonic carcinoma (EC) stem cells, NTERA2 (NT2)
which highly expressed SOX2OT and SOX2.
SOX2OT
0.0012
0.001
0.0008
0.0006
0.0004
0.0002
0
1
2
3
Week
4
0.0005
0.00045
0.0004
0.00035
0.0003
0.00025
0.0002
0.00015
0.0001
0.00005
0
SOX2DOT
1
2
3
4
Week
Figure4: Different expression patterns of SOX2OT and SOX2DOT during the
process of neural differentiation of NT2 stem cells
Conclusion
All together, our data provide a novel regulatory mechanism governing one
of key stem cell pluripotency genes, SOX2, mediated by the lncRNA SOX2OT.
30
20
10
0
G1
S
G2/M
Figure2: SOX2OT gene silencing caused the loss of SOX2 expression (0.1,
P< 0.01) (left) and caused G1 cell cycle arrest in NT2 cells (right).
References
1- Sheik Mohamed J et al. Conserved long noncoding RNAs transcriptionally
regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic
stem cells. RNA 2010; 16: 324–337
2- Fantes J et al. Mutations in SOX2 cause anophthalmia. Nature genetics
2003; 33, 461-3.
3- Amaral PP et al. Complex architecture and regulated expression of the
Sox2ot locus during vertebrate
development. RNA 2009; 15, 2013-27.
*To whom Correspondence should be addressed: Seyed Javad Mowla Ph.D.,
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat
Modares University, Tehran, Iran E-mail: [email protected]