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The NKG2D ligands RAE-1δ and RAE-1ε differ with respect to their receptor affinity, expression profiles and transcriptional regulation.

Cédile O, Popa N, Pollet-Villard F, Garmy N, Ibrahim EC, Boucraut J - PLoS ONE (2010)

Bottom Line: The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε.Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression.Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

View Article: PubMed Central - PubMed

Affiliation: CRN2M, CNRS UMR 6231, Université de la Méditerranée, Université Paul Cézanne, Faculté de Médecine, Marseille, France.

ABSTRACT

Background: RAE-1 is a ligand of the activating receptor NKG2D expressed by NK cells, NKT, γδT and some CD8(+)T lymphocytes. RAE-1 is overexpressed in tumor cell lines and its expression is induced after viral infection and genotoxic stress. We have recently demonstrated that RAE-1 is expressed in the adult subventricular zone (SVZ) from C57BL/6 mice. RAE-1 is also expressed in vitro by neural stem/progenitor cells (NSPCs) and plays a non-immune role in cell proliferation. The C57BL/6 mouse genome contains two rae-1 genes, rae-1δ and rae-1ε encoding two different proteins. The goals of this study are first to characterize the in vivo and in vitro expression of each gene and secondly to elucidate the mechanisms underlying their respective expression, which are far from known.

Principal findings: We observed that Rae-1δ and Rae-1ε transcripts are differentially expressed according to tissues, pathological conditions and cell lines. Embryonic tissue and the adult SVZ mainly expressed Rae-1δ transcripts. The NSPCs derived from the SVZ also mainly expressed RAE-1δ. The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε. On the contrary, cell lines expressed either similar levels of RAE-1δ and RAE-1ε proteins or only RAE-1ε. Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression. We indeed identified two different promoter regions for each gene: one mainly involved in the control of rae-1δ gene expression and the other in the control of rae-1ε expression.

Conclusions/significance: RAE-1δ and RAE-1ε differ with respect to their function and the control of their expression. Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

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Characterization of Rae-1δ and Rae-1ε transcripts.A, RT-PCR analysis of Rae-1δ transcripts using an exon 1-exon 6δ primer pair or an exon 5-exon 6δ primer pair and of Rae-1ε transcripts using an exon 1-exon 6ε primer pair or an exon 5-exon 6ε primer pair (see schematic in Fig. 6). We compared the PCR products obtained from cell lines (C57sv, ES, NOE) and healthy tissues (10-days-old embryo (E10), liver, heart (He) and brain (Br)); NTC, non template control.
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pone-0013466-g004: Characterization of Rae-1δ and Rae-1ε transcripts.A, RT-PCR analysis of Rae-1δ transcripts using an exon 1-exon 6δ primer pair or an exon 5-exon 6δ primer pair and of Rae-1ε transcripts using an exon 1-exon 6ε primer pair or an exon 5-exon 6ε primer pair (see schematic in Fig. 6). We compared the PCR products obtained from cell lines (C57sv, ES, NOE) and healthy tissues (10-days-old embryo (E10), liver, heart (He) and brain (Br)); NTC, non template control.

Mentions: We then analyzed all Rae-1 transcripts possibly generated by these promoters in C57BL/6 cell lines and tissues using RT-PCR and agarose gel analysis. The Ex5-Ex9 primer pair does not discriminate the Rae-1δ from the Rae-1ε transcripts (data not shown). Using these primers, we detected a single product. We sequenced the liver PCR product obtained with those primers and observed the superimposition of two sequences, one corresponding to Rae-1δ and the other one less represented corresponding to Rae-1ε (Figure S1). The figure 4 illustrates the results obtained with Ex1-Ex6δ and Ex1-Ex6ε pairs that are gene-specific and may reflect the activity of the putative promoter upstream of exon 1. Using an exon 5 and exon 6δ primer pair, Rae-1δ transcripts were detected in all tested conditions. However, we failed to detect Rae-1δ transcripts using the primer targeting exon 1 (Fig. 4, upper panels). We only detected exon 1-containing Rae-1δ transcripts in the NOE cell line (referenced FJ594066 in GenBank) after treatment with actinomycin D and cycloheximide which stabilize RNA (data not shown). Finally, the primer pair targeting exon 1 and exon 6 of Rae-1ε, allowed the detection of three main PCR products, one full length and two alternative splicing products of either exon 2 alone (402 bp) or exon 2 and exon 3 (336 bp) (Fig. 4, middle panels) which were sequenced and referenced in GenBank, FJ 594065, FJ594067 and FJ594068 respectively. These observations suggested that transcription of rae-1 genes is under the control of two different promoter regions, one upstream of exon 1 controlling the expression of Rae-1ε, and the second, upstream of exon 5 mainly involved in the expression of Rae-1δ.


The NKG2D ligands RAE-1δ and RAE-1ε differ with respect to their receptor affinity, expression profiles and transcriptional regulation.

Cédile O, Popa N, Pollet-Villard F, Garmy N, Ibrahim EC, Boucraut J - PLoS ONE (2010)

Characterization of Rae-1δ and Rae-1ε transcripts.A, RT-PCR analysis of Rae-1δ transcripts using an exon 1-exon 6δ primer pair or an exon 5-exon 6δ primer pair and of Rae-1ε transcripts using an exon 1-exon 6ε primer pair or an exon 5-exon 6ε primer pair (see schematic in Fig. 6). We compared the PCR products obtained from cell lines (C57sv, ES, NOE) and healthy tissues (10-days-old embryo (E10), liver, heart (He) and brain (Br)); NTC, non template control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957426&req=5

pone-0013466-g004: Characterization of Rae-1δ and Rae-1ε transcripts.A, RT-PCR analysis of Rae-1δ transcripts using an exon 1-exon 6δ primer pair or an exon 5-exon 6δ primer pair and of Rae-1ε transcripts using an exon 1-exon 6ε primer pair or an exon 5-exon 6ε primer pair (see schematic in Fig. 6). We compared the PCR products obtained from cell lines (C57sv, ES, NOE) and healthy tissues (10-days-old embryo (E10), liver, heart (He) and brain (Br)); NTC, non template control.
Mentions: We then analyzed all Rae-1 transcripts possibly generated by these promoters in C57BL/6 cell lines and tissues using RT-PCR and agarose gel analysis. The Ex5-Ex9 primer pair does not discriminate the Rae-1δ from the Rae-1ε transcripts (data not shown). Using these primers, we detected a single product. We sequenced the liver PCR product obtained with those primers and observed the superimposition of two sequences, one corresponding to Rae-1δ and the other one less represented corresponding to Rae-1ε (Figure S1). The figure 4 illustrates the results obtained with Ex1-Ex6δ and Ex1-Ex6ε pairs that are gene-specific and may reflect the activity of the putative promoter upstream of exon 1. Using an exon 5 and exon 6δ primer pair, Rae-1δ transcripts were detected in all tested conditions. However, we failed to detect Rae-1δ transcripts using the primer targeting exon 1 (Fig. 4, upper panels). We only detected exon 1-containing Rae-1δ transcripts in the NOE cell line (referenced FJ594066 in GenBank) after treatment with actinomycin D and cycloheximide which stabilize RNA (data not shown). Finally, the primer pair targeting exon 1 and exon 6 of Rae-1ε, allowed the detection of three main PCR products, one full length and two alternative splicing products of either exon 2 alone (402 bp) or exon 2 and exon 3 (336 bp) (Fig. 4, middle panels) which were sequenced and referenced in GenBank, FJ 594065, FJ594067 and FJ594068 respectively. These observations suggested that transcription of rae-1 genes is under the control of two different promoter regions, one upstream of exon 1 controlling the expression of Rae-1ε, and the second, upstream of exon 5 mainly involved in the expression of Rae-1δ.

Bottom Line: The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε.Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression.Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

View Article: PubMed Central - PubMed

Affiliation: CRN2M, CNRS UMR 6231, Université de la Méditerranée, Université Paul Cézanne, Faculté de Médecine, Marseille, France.

ABSTRACT

Background: RAE-1 is a ligand of the activating receptor NKG2D expressed by NK cells, NKT, γδT and some CD8(+)T lymphocytes. RAE-1 is overexpressed in tumor cell lines and its expression is induced after viral infection and genotoxic stress. We have recently demonstrated that RAE-1 is expressed in the adult subventricular zone (SVZ) from C57BL/6 mice. RAE-1 is also expressed in vitro by neural stem/progenitor cells (NSPCs) and plays a non-immune role in cell proliferation. The C57BL/6 mouse genome contains two rae-1 genes, rae-1δ and rae-1ε encoding two different proteins. The goals of this study are first to characterize the in vivo and in vitro expression of each gene and secondly to elucidate the mechanisms underlying their respective expression, which are far from known.

Principal findings: We observed that Rae-1δ and Rae-1ε transcripts are differentially expressed according to tissues, pathological conditions and cell lines. Embryonic tissue and the adult SVZ mainly expressed Rae-1δ transcripts. The NSPCs derived from the SVZ also mainly expressed RAE-1δ. The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε. On the contrary, cell lines expressed either similar levels of RAE-1δ and RAE-1ε proteins or only RAE-1ε. Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression. We indeed identified two different promoter regions for each gene: one mainly involved in the control of rae-1δ gene expression and the other in the control of rae-1ε expression.

Conclusions/significance: RAE-1δ and RAE-1ε differ with respect to their function and the control of their expression. Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

Show MeSH
Related in: MedlinePlus