Dataset Information

Identification and Characterization of a Splicing Variant in the 5' UTR of the Human TLR5 Gene.

ABSTRACT: Toll-like receptors (TLRs) are essential components of the innate immune system. TLR5 is the receptor for flagellin, the principal protein component of bacterial flagella. The TLR5 gene has 6 exons. In an RT-PCR analysis, we found long TLR5 transcripts, in addition to those of the expected size (short TLR5 transcripts). A sequence analysis revealed that the long TLR5 transcripts contain a new exon of 94 nucleotides located between previously reported exons IV and V in the 5' untranslated region (5' UTR). A real-time PCR analysis of the two alternatively spliced variants in various cell lines showed that the long TLR5 transcripts are abundantly expressed in nonimmune cells. The ratios of long/short transcripts in human nonimmune cell lines, such as A549, T98G, HaCaT, H460, HEK-293, and Caco-2 cells, and primary mesenchymal stem cells were in the range of 1.25 to 4.31. In contrast, those of human monocytic THP-1 and U937 cells and E6.1 T cells and Ramos B cells were around 0.9. These ratios in human monocytic THP-1 cells were decreased by treatment with IFN-? in a concentration-dependent manner. Based on our findings, we suggest that the newly found long TLR5 transcripts may be involved in the negative regulation of TLR5 expression and function.

SUBMITTER: Hoang TX

PROVIDER: S-EPMC5602658 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Identification and Characterization of a Splicing Variant in the 5' UTR of the Human TLR5 Gene.

Hoang Thi Xoan TX Duong Cao Nguyen CN Kim Jae Young JY

BioMed research international 20170829

Toll-like receptors (TLRs) are essential components of the innate immune system. TLR5 is the receptor for flagellin, the principal protein component of bacterial flagella. The TLR5 gene has 6 exons. In an RT-PCR analysis, we found long TLR5 transcripts, in addition to those of the expected size (short TLR5 transcripts). A sequence analysis revealed that the long TLR5 transcripts contain a new exon of 94 nucleotides located between previously reported exons IV and V in ...[more]

PMID: 28948171

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Project description:DJ-1 forms part of the neuronal cellular defence mechanism against oxidative insults, due to its ability to undergo self-oxidation. Oxidative stress has been implicated in the pathogenesis of central nervous system damage in different neurodegenerative disorders including Alzheimer's disease and Parkinson's disease (PD). Various mutations in the DJ-1 (PARK7) gene have been shown to cause the autosomal recessive form of PD. In the present study South African PD patients were screened for mutations in DJ-1 and we aimed to investigate the functional significance of a novel 16 bp deletion variant identified in one patient.The possible effect of the deletion on promoter activity was investigated using a Dual-Luciferase Reporter assay. The DJ-1 5'-UTR region containing the sequence flanking the 16 bp deletion was cloned into a pGL4.10-Basic luciferase-reporter vector and transfected into HEK293 and BE(2)-M17 neuroblastoma cells. Promoter activity under hydrogen peroxide-induced oxidative stress conditions was also investigated. Computational (in silico) cis-regulatory analysis of DJ-1 promoter sequence was performed using the transcription factor-binding site database, TRANSFAC via the PATCH and rVISTA platforms.A novel 16 bp deletion variant (g.-6_+10del) was identified in DJ-1 which spans the transcription start site and is situated 93 bp 3' from a Sp1 site. The deletion caused a reduction in luciferase activity of approximately 47% in HEK293 cells and 60% in BE(2)-M17 cells compared to the wild-type (P < 0.0001), indicating the importance of the 16 bp sequence in transcription regulation. The activity of both constructs was up-regulated during oxidative stress. Bioinformatic analysis revealed putative binding sites for three transcription factors AhR, ARNT, HIF-1 within the 16 bp sequence. The frequency of the g.-6_+10del variant was determined to be 0.7% in South African PD patients (2 heterozygotes in 148 individuals).This is the first report of a functional DJ-1 promoter variant, which has the potential to influence transcript stability or translation efficiency. Further work is necessary to determine the extent to which the g.-6_+10del variant affects the normal function of the DJ-1 promoter and whether this variant confers a risk for PD.

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Dataset Information

Identification and Characterization of a Splicing Variant in the 5' UTR of the Human TLR5 Gene.

Publications

Identification and Characterization of a Splicing Variant in the 5' UTR of the Human <i>TLR5</i> Gene.

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