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Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

Presnell SR, Al-Attar A, Cichocki F, Miller JS, Lutz CT - Genes Immun. (2014)

Bottom Line: We found that few microRNAs (miRNAs) differed significantly between human NK and T cells.TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1.The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

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Primary miR-181ab-1 and -2 transcript mapping. Purified NK cells of three subjects were cultured overnight with culture media (A-C) or with TGF-β (D). Following reverse transcription, qPCR was performed using primers with sequences homologous to genomic DNA at the location shown by figure data points. For each subject, determination of relative RNA levels compared to the “middle primers” located between the miR-181a and miR-181b coding sequences (arrows) were calculated as: fold difference = 2 (Cq middle primer - Cq specific primer). When no amplification was detected, the Cq was set to 40.0. The Avg and SEM are presented on a log10 scale; some error bars are too small to be seen. The numbers in each graph indicate genomic position. In A, for example, “825” indicates Chromosome 1:198 825 000. In C, “450” represents Chromosome 9:127 450 000. A, B. MIR181A1B1. B. An expanded view of Chromosome 1:198 826 100-198 835 200, which highlights the region near pre-miR-181a. C, D. MIR181A2B2. The amount of pri-miR-181ab-2 RNA in NK cells incubated in culture media (C) or with TGF-β (20 ng/mL, D).
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Figure 4: Primary miR-181ab-1 and -2 transcript mapping. Purified NK cells of three subjects were cultured overnight with culture media (A-C) or with TGF-β (D). Following reverse transcription, qPCR was performed using primers with sequences homologous to genomic DNA at the location shown by figure data points. For each subject, determination of relative RNA levels compared to the “middle primers” located between the miR-181a and miR-181b coding sequences (arrows) were calculated as: fold difference = 2 (Cq middle primer - Cq specific primer). When no amplification was detected, the Cq was set to 40.0. The Avg and SEM are presented on a log10 scale; some error bars are too small to be seen. The numbers in each graph indicate genomic position. In A, for example, “825” indicates Chromosome 1:198 825 000. In C, “450” represents Chromosome 9:127 450 000. A, B. MIR181A1B1. B. An expanded view of Chromosome 1:198 826 100-198 835 200, which highlights the region near pre-miR-181a. C, D. MIR181A2B2. The amount of pri-miR-181ab-2 RNA in NK cells incubated in culture media (C) or with TGF-β (20 ng/mL, D).

Mentions: The MIR181A1B1 and MIR181A2B2 transcriptional units and their promoters have not been established, so we first performed RT-qPCR at intervals upstream and downstream of the mature miR-181 sequences to measure the extent of the MIR181ab transcriptional units. Fig. 4 shows that RT-qPCR signals were relatively constant for several kb upstream and downstream of the mature miR-181a/b sequences on both chromosomes 1 and 9 in human NK cells. Variations in RT-qPCR signal strength may be due to unequal RNA degradation rates, differences in priming efficiency, or RNA secondary structure formation affecting reverse transcription efficiency. Some unspliced pri-miRNAs are present at higher levels than their spliced counterparts 25 and the relative constancy of pri-miR-181 levels across the transcriptional units suggests that the major forms are unspliced transcripts. Levels of pri-miR-181ab-1 gradually fell 67-fold in the interval 2-49 kb downstream of the mature miRNA sequences (Fig. 4A). This gradual decline suggests the absence of a single dominant RNA polymerase II termination site in MIR181A1B1. In contrast, pri-miR-181ab-2 levels fell > 50-fold somewhere in the interval 21.5-29 kb downstream of the mature miR-181a/b-2 coding sequences (Fig. 4C), consistent with a dominant MIR181A2B2 RNA polymerase II termination site.


Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

Presnell SR, Al-Attar A, Cichocki F, Miller JS, Lutz CT - Genes Immun. (2014)

Primary miR-181ab-1 and -2 transcript mapping. Purified NK cells of three subjects were cultured overnight with culture media (A-C) or with TGF-β (D). Following reverse transcription, qPCR was performed using primers with sequences homologous to genomic DNA at the location shown by figure data points. For each subject, determination of relative RNA levels compared to the “middle primers” located between the miR-181a and miR-181b coding sequences (arrows) were calculated as: fold difference = 2 (Cq middle primer - Cq specific primer). When no amplification was detected, the Cq was set to 40.0. The Avg and SEM are presented on a log10 scale; some error bars are too small to be seen. The numbers in each graph indicate genomic position. In A, for example, “825” indicates Chromosome 1:198 825 000. In C, “450” represents Chromosome 9:127 450 000. A, B. MIR181A1B1. B. An expanded view of Chromosome 1:198 826 100-198 835 200, which highlights the region near pre-miR-181a. C, D. MIR181A2B2. The amount of pri-miR-181ab-2 RNA in NK cells incubated in culture media (C) or with TGF-β (20 ng/mL, D).
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Figure 4: Primary miR-181ab-1 and -2 transcript mapping. Purified NK cells of three subjects were cultured overnight with culture media (A-C) or with TGF-β (D). Following reverse transcription, qPCR was performed using primers with sequences homologous to genomic DNA at the location shown by figure data points. For each subject, determination of relative RNA levels compared to the “middle primers” located between the miR-181a and miR-181b coding sequences (arrows) were calculated as: fold difference = 2 (Cq middle primer - Cq specific primer). When no amplification was detected, the Cq was set to 40.0. The Avg and SEM are presented on a log10 scale; some error bars are too small to be seen. The numbers in each graph indicate genomic position. In A, for example, “825” indicates Chromosome 1:198 825 000. In C, “450” represents Chromosome 9:127 450 000. A, B. MIR181A1B1. B. An expanded view of Chromosome 1:198 826 100-198 835 200, which highlights the region near pre-miR-181a. C, D. MIR181A2B2. The amount of pri-miR-181ab-2 RNA in NK cells incubated in culture media (C) or with TGF-β (20 ng/mL, D).
Mentions: The MIR181A1B1 and MIR181A2B2 transcriptional units and their promoters have not been established, so we first performed RT-qPCR at intervals upstream and downstream of the mature miR-181 sequences to measure the extent of the MIR181ab transcriptional units. Fig. 4 shows that RT-qPCR signals were relatively constant for several kb upstream and downstream of the mature miR-181a/b sequences on both chromosomes 1 and 9 in human NK cells. Variations in RT-qPCR signal strength may be due to unequal RNA degradation rates, differences in priming efficiency, or RNA secondary structure formation affecting reverse transcription efficiency. Some unspliced pri-miRNAs are present at higher levels than their spliced counterparts 25 and the relative constancy of pri-miR-181 levels across the transcriptional units suggests that the major forms are unspliced transcripts. Levels of pri-miR-181ab-1 gradually fell 67-fold in the interval 2-49 kb downstream of the mature miRNA sequences (Fig. 4A). This gradual decline suggests the absence of a single dominant RNA polymerase II termination site in MIR181A1B1. In contrast, pri-miR-181ab-2 levels fell > 50-fold somewhere in the interval 21.5-29 kb downstream of the mature miR-181a/b-2 coding sequences (Fig. 4C), consistent with a dominant MIR181A2B2 RNA polymerase II termination site.

Bottom Line: We found that few microRNAs (miRNAs) differed significantly between human NK and T cells.TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1.The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

Show MeSH
Related in: MedlinePlus