Novel noncoding antisense RNA transcribed from human anti-NOS2A locus is differentially regulated during neuronal differentiation of embryonic stem cells - PubMed (original) (raw)

Novel noncoding antisense RNA transcribed from human anti-NOS2A locus is differentially regulated during neuronal differentiation of embryonic stem cells

Sergei A Korneev et al. RNA. 2008 Oct.

Abstract

Here, we report on the discovery of a locus in the human genome, which evolved by gene duplication followed by an internal DNA inversion. This locus exhibits high sequence similarity to the gene for the inducible isoform of NOS protein (NOS2A) and is transcribed into a noncoding RNA containing a region of significant antisense homology with the NOS2A mRNA. We show that this antisense transcript (anti-NOS2A RNA) is expressed in different types of brain tumors, including meningiomas and glioblastomas. More importantly, we demonstrate that the expression profiles of the anti-NOS2A RNA and the NOS2A mRNA exhibit concurrent reciprocal changes in undifferentiated human embryonic stem cells (hESCs) and in hESCs induced to differentiate into neurogenic precursors such as neurospheres. As NOS2A has a role in neurogenesis, our results suggest that the anti-NOS2A RNA is involved in the regulation of neuronal differentiation of hESCs through the modulation of NOS2A gene expression.

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Figures

FIGURE 1.

FIGURE 1.

Evolution of anti-NOS2A locus. (A) The chain of evolutionary events suggested by our studies and leading to the creation of the anti-NOS2A locus in the human genome involved partial duplication (indicated by the linked arrows) of an ancestral NOS2A gene coupled to an internal DNA inversion (hatched box) within the duplicated copy (light gray box). Because of such dramatic disruption of the organization, the anti-NOS2A locus cannot produce mature NOS2A mRNA. Instead, it can be transcribed into a NOS2A-related _trans_NAT. (B) Organization of orthologous anti-NOS2A loci in the human (Homo sapiens), orangutan (Pongo pygmaeus), chimpanzee (Pan troglodytes), and rhesus macaque (Macaca mulatta) genomes. Internal DNA inversions are shown by hatched boxes.

FIGURE 2.

FIGURE 2.

Anti-NOS2A locus is transcribed into noncoding NOS2A-related NAT. (A) Structural organization of human anti-NOS2A locus. The inverted part of the locus is shown by the hatched box. Primers used in RT-PCR experiments are numbered. The DNA sequence of the anti-NOS2A locus has been analyzed using Promoter 2.0 software (Knudsen 1999). Notably, a region located within the inverted segment has scored 1.082, indicating the very high likelihood of the presence of a functional promoter. The promoter was predicted to drive transcription of the anti-NOS2A locus in the opposite orientation with respect to NOS2A gene. The putative transcription start site and the direction of transcription are shown by an arrow. A DNA region, transcription of which results in the synthesis of RNA fragment antisense to the NOS2A mRNA, is indicated in black. (B) The results of RT-PCR experiments performed on total RNA extracted from human meningioma (lanes 1,2) and glioblastoma (lane 3) using primers 1 and 2 (lanes 1,3) or primers 3 and 4 (lane 2). (C) The results of RT-PCR experiments performed on total RNA from human meningioma using primers 1 and 2. A sequence-specific primer complimentary to a putative RNA containing NOS2A mRNA-homologous region in antisense orientation was used in the RT reaction. Note that there is no product in the experiments in which reverse transcriptase was omitted from the first strand synthesis reaction (lane 2). (D) The results of RT-PCR experiments performed on total RNA from human meningioma using primers 1 and 2 and either poly(A)+ (lanes 1,3) or poly(A)− (lanes 2,4) RNA fractions. Note that there are no products in the experiments in which reverse transcriptase was omitted from the first-strand synthesis reactions (lanes 3,4). (E) Alignment of the antisense region of the anti-NOS2A RNA with its complementary counterpart in the NOS2A mRNA. In this alignment there is >80% complementarity.

FIGURE 3.

FIGURE 3.

The results of real-time RT-PCR performed on hESCs. (A) Colonies of hESM01 on the feeder layer of mouse embryonic fibroblasts. Scale bar represents 200 μm. (B) Neurospheres derived from hESM01 grown in suspension for 1 wk. Scale bar represents 200 μm. (C) Immunohistochemical analysis of differentiating neurosphere derived from hESM01. Cells were stained with antibodies against human NSE (green) and GFAP (red). Scale bar represents 200 μm. (D, E) Circular diagrams illustrating the ratio between relative levels of NOS2A mRNA (gray sectors) and anti-NOS2A RNA (white sectors) expression in undifferentiated hESM01 (D) and in neurospheres (E) derived from hESM01. The ratios were calculated as formula image.

FIGURE 4.

FIGURE 4.

The expression profiles of the anti-NOS2A RNA and the NOS2A mRNA exhibit concurrent reciprocal changes in undifferentiated hESCs and in hESCs induced to differentiate into neurospheres. (A, B) The relative levels of anti-NOS2A RNA (A) and NOS2A mRNA (B) expression normalized to an endogenous control (GAPDH) and relative to a calibrator in undifferentiated hESM01 (dark gray bars) and in neurospheres derived from hESM01 (light gray bars).

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