MxA transcripts with distinct first exons and modulation of gene expression levels by single-nucleotide polymorphisms in human bronchial epithelial cells

Abstract

Myxovirus resistance A (MxA) is a major interferon (IFN)-inducible antiviral protein. Promoter single-nucleotide polymorphisms (SNPs) of MxA near the IFN-stimulated response element (ISRE) have been frequently associated with various viral diseases, including emerging respiratory infections. We investigated the expression profile of MxA transcripts with distinct first exons in human bronchial epithelial cells. For primary culture, the bronchial epithelium was isolated from lung tissues with different genotypes, and total RNA was subjected to real-time reverse transcription polymerase chain reaction. The previously reported MxA transcript (T1) and a recently registered transcript with a distinct 5' first exon (T0) were identified. IFN-β and polyinosinic-polycytidylic acid induced approximately 100-fold higher expression of the T1 transcript than that of the T0 transcript, which also had a potential ISRE motif near its transcription start site. Even without inducers, the T1 transcript accounted for approximately two thirds of the total expression of MxA, levels of which were significantly associated with its promoter and exon 1 SNPs (rs17000900, rs2071430, and rs464138). Our results suggest that MxA observed in respiratory viral infections is possibly dominated by the T1 transcript and partly influenced by relevant 5' SNPs.

Fig. 1

Alternate splicing of 5′ exons in MxA. The 5′ genomic structure of MxA and nucleotide sequences around the transcription start sites of the T0 and T1 transcripts are shown. White boxes represent the untranslated mRNA sequence, and black boxes represent the translated sequence. Potential ISREs are double underlined, NF-κB binding site is underlined, and promoter and exon 1 SNPs are boxed. The transcription start site (Horisberger et al. 1990) and nucleotide positions shown in the T1 transcript are displayed in accordance with MxA promoter analysis by Ronni et al. (1998)

Fig. 2

Relative expression levels of two MxA transcript variants in human tissues. Relative expression levels of the T0 and T1 transcripts in various human tissues were obtained by real-time RT-PCR using a commercial RNA panel, Human Total RNA Master panel II (Clontech). The RNA consisted of pooled RNA from two or more donors. Their genotypes were not available but presumably mixture of different genotypes

Fig. 3

Induction of T0 and T1 transcript variants by various stimuli in HBE cells. HBE cells (n = 3) were stimulated with IFN-α, IFN-β, poly(I:C), IFN-γ, TNF-α, LPS, α-defensin 1, β-defensin 1, and β-defensin 2 for 24 h and then harvested. Expression levels of the T0 and T1 transcripts were compared with those of unstimulated cells by real-time RT-PCR. Fold inductions are shown as the mean ± SEM. The genotypes of the promoter SNPs were as follows: sample #1, −77 SNP (rs457274) C/G, −123 SNP (rs17000900) C/A, −88 SNP (rs2071430) G/T, and +20 SNP (rs464138) C/A; sample #2, −77 C/C, −123 C/A, −88 T/T, and +20 A/A and; sample #3, −77 C/G, −123 C/C, −88 G/G, and +20 C/A

Fig. 4

Differences in the baseline expression of transcript variants among the genotypes of the promoter and exon 1 SNPs in HBE cells. Expression of a T0 and b T1 transcripts under the unstimulated condition in HBE cells with each genotype of −77 C/G SNP for the T0 transcript (C/C, n = 8; C/G, n = 18; G/G, n = 12), of −123 C/A SNP (A/A, n = 2; A/C, n = 18; C/C, n = 18), of −88 G/T SNP (T/T, n = 3; G/T, n = 19; G/G, n = 16), and of +20 C/A SNP (A/A, n = 5; A/C, n = 22; C/C, n = 11) for the T1 transcript is shown. The relative amounts of mRNA of each transcript compared with that of the T0 transcript in GG cells without poly(I:C) stimulation are shown as mean ± SEM. Possible associations between the number of alleles and the amount of the corresponding transcripts were assessed by a simple regression model respectively (p = 0.086 for the number of −77 C alleles, p = 0.013 for −123 A alleles, p = 0.0035 for −88 T alleles, and p < 0.0001 for +20 A alleles)

Fig. 5

Differences in the poly(I:C)-induced expression of transcript variants among the genotypes of the promoter and exon 1 SNPs in HBE cells. Expression of a T0 and b T1 transcripts in the presence of poly(I:C) in HBE cells with each genotype of −77 C/G SNP for the T0 transcript (C/C, n = 5; C/G, n = 13; G/G, n = 11), of −123 C/A SNP (A/A, n = 1; A/C, n = 12; C/C, n = 16), of −88 G/T SNP (T/T, n = 2; G/T, n = 13; G/G, n = 14), and of +20 C/A SNP (A/A, n = 3; A/C, n = 16; C/C, n = 10) for the T1 transcript is shown. The relative amounts of mRNA of each transcript compared with that of the T0 transcript in GG cells without poly(I:C) stimulation are shown as mean ± SEM. Possible associations were assessed by a simple regression model (p = 0.647 for the number of −77 C alleles, p = 0.662 for −123 A alleles, p = 0.539 for −88 T alleles, and p = 0.331 for +20 A alleles)