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Aire Downregulation Is Associated with Changes in the Posttranscriptional Control of Peripheral Tissue Antigens in Medullary Thymic Epithelial Cells

View Article: PubMed Central - PubMed

ABSTRACT

Autoimmune regulator (Aire) is a transcriptional regulator of peripheral tissue antigens (PTAs) and microRNAs (miRNAs) in medullary thymic epithelial cells (mTECs). In this study, we tested the hypothesis that Aire also played a role as an upstream posttranscriptional controller in these cells and that variation in its expression might be associated with changes in the interactions between miRNAs and the mRNAs encoding PTAs. We demonstrated that downregulation of Aire in vivo in the thymuses of BALB/c mice imbalanced the large-scale expression of these two RNA species and consequently their interactions. The expression profiles of a large set of mTEC miRNAs and mRNAs isolated from the thymuses of mice subjected (or not) to small-interfering-induced Aire gene knockdown revealed that 87 miRNAs and 4,558 mRNAs were differentially expressed. The reconstruction of the miRNA–mRNA interaction networks demonstrated that interactions between these RNAs were under Aire influence and therefore changed when this gene was downregulated. Prior to Aire-knockdown, only members of the miR-let-7 family interacted with a set of PTA mRNAs. Under Aire-knockdown conditions, a larger set of miRNA families and their members established this type of interaction. Notably, no previously described Aire-dependent PTA interacted with the miRNAs, indicating that these PTAs were somehow refractory. The miRNA–mRNA interactions were validated by calculating the minimal free energy of the pairings between the miRNA seed regions and the mRNA 3′ UTRs and within the cellular milieu using the luciferase reporter gene assay. These results suggest the existence of a link between transcriptional and posttranscriptional control because Aire downregulation alters the miRNA–mRNA network controlling PTAs in mTEC cells.

No MeSH data available.


Immunofluorescence for Aire protein 48 h after anti-Aire siRNA electro-transfection. (A) Thymus electro-transfected with PBS buffer (control) or (B) electro-transfected with anti-Aire siRNA. Nuclei of cells were stained with DAPI (blue) and for AIRE protein in green by using goat anti-Aire IgG primary antibody and fluorescein-conjugated rabbit anti-goat IgG secondary antibody. Comparing to the control thymus, immunofluorescence signal for AIRE protein was strikingly reduced in the nuclei of cells of Aire-knockdown thymus. Magnification 40×, c, cortex; m, medulla.
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Figure 2: Immunofluorescence for Aire protein 48 h after anti-Aire siRNA electro-transfection. (A) Thymus electro-transfected with PBS buffer (control) or (B) electro-transfected with anti-Aire siRNA. Nuclei of cells were stained with DAPI (blue) and for AIRE protein in green by using goat anti-Aire IgG primary antibody and fluorescein-conjugated rabbit anti-goat IgG secondary antibody. Comparing to the control thymus, immunofluorescence signal for AIRE protein was strikingly reduced in the nuclei of cells of Aire-knockdown thymus. Magnification 40×, c, cortex; m, medulla.

Mentions: After demonstrating by RT-qPCR that the in vivo anti-Aire siRNA electro-transfection was an effective method to reduce Aire mRNA expression in the thymus, immunofluorescence microscopy was used to detect the AIRE protein in thymus tissue sections under Aire-knockdown conditions. Figure 2 shows the detection of this protein (green fluorescence) exclusively in cells of the thymic medullary compartment. The thymus under Aire-knockdown conditions featured a reduction in the number of Aire+ cells and in the intensity of the fluorescence signal, which reflected a reduction in AIRE protein expression. Moreover, immunoblotting was used to measure the AIRE protein levels under the Aire-knockdown conditions. Figure S3 in Supplementary Material demonstrates the detection of the 55 kDa AIRE protein in the control thymus and an approximately 50% reduction of this protein in the Aire-knockdown thymus.


Aire Downregulation Is Associated with Changes in the Posttranscriptional Control of Peripheral Tissue Antigens in Medullary Thymic Epithelial Cells
Immunofluorescence for Aire protein 48 h after anti-Aire siRNA electro-transfection. (A) Thymus electro-transfected with PBS buffer (control) or (B) electro-transfected with anti-Aire siRNA. Nuclei of cells were stained with DAPI (blue) and for AIRE protein in green by using goat anti-Aire IgG primary antibody and fluorescein-conjugated rabbit anti-goat IgG secondary antibody. Comparing to the control thymus, immunofluorescence signal for AIRE protein was strikingly reduced in the nuclei of cells of Aire-knockdown thymus. Magnification 40×, c, cortex; m, medulla.
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Related In: Results  -  Collection

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Figure 2: Immunofluorescence for Aire protein 48 h after anti-Aire siRNA electro-transfection. (A) Thymus electro-transfected with PBS buffer (control) or (B) electro-transfected with anti-Aire siRNA. Nuclei of cells were stained with DAPI (blue) and for AIRE protein in green by using goat anti-Aire IgG primary antibody and fluorescein-conjugated rabbit anti-goat IgG secondary antibody. Comparing to the control thymus, immunofluorescence signal for AIRE protein was strikingly reduced in the nuclei of cells of Aire-knockdown thymus. Magnification 40×, c, cortex; m, medulla.
Mentions: After demonstrating by RT-qPCR that the in vivo anti-Aire siRNA electro-transfection was an effective method to reduce Aire mRNA expression in the thymus, immunofluorescence microscopy was used to detect the AIRE protein in thymus tissue sections under Aire-knockdown conditions. Figure 2 shows the detection of this protein (green fluorescence) exclusively in cells of the thymic medullary compartment. The thymus under Aire-knockdown conditions featured a reduction in the number of Aire+ cells and in the intensity of the fluorescence signal, which reflected a reduction in AIRE protein expression. Moreover, immunoblotting was used to measure the AIRE protein levels under the Aire-knockdown conditions. Figure S3 in Supplementary Material demonstrates the detection of the 55 kDa AIRE protein in the control thymus and an approximately 50% reduction of this protein in the Aire-knockdown thymus.

View Article: PubMed Central - PubMed

ABSTRACT

Autoimmune regulator (Aire) is a transcriptional regulator of peripheral tissue antigens (PTAs) and microRNAs (miRNAs) in medullary thymic epithelial cells (mTECs). In this study, we tested the hypothesis that Aire also played a role as an upstream posttranscriptional controller in these cells and that variation in its expression might be associated with changes in the interactions between miRNAs and the mRNAs encoding PTAs. We demonstrated that downregulation of Aire in vivo in the thymuses of BALB/c mice imbalanced the large-scale expression of these two RNA species and consequently their interactions. The expression profiles of a large set of mTEC miRNAs and mRNAs isolated from the thymuses of mice subjected (or not) to small-interfering-induced Aire gene knockdown revealed that 87 miRNAs and 4,558 mRNAs were differentially expressed. The reconstruction of the miRNA–mRNA interaction networks demonstrated that interactions between these RNAs were under Aire influence and therefore changed when this gene was downregulated. Prior to Aire-knockdown, only members of the miR-let-7 family interacted with a set of PTA mRNAs. Under Aire-knockdown conditions, a larger set of miRNA families and their members established this type of interaction. Notably, no previously described Aire-dependent PTA interacted with the miRNAs, indicating that these PTAs were somehow refractory. The miRNA–mRNA interactions were validated by calculating the minimal free energy of the pairings between the miRNA seed regions and the mRNA 3′ UTRs and within the cellular milieu using the luciferase reporter gene assay. These results suggest the existence of a link between transcriptional and posttranscriptional control because Aire downregulation alters the miRNA–mRNA network controlling PTAs in mTEC cells.

No MeSH data available.