Abstract
Mediator (MED) is a key regulator of protein-coding gene expression, and mutations in MED subunits are associated with a broad spectrum of diseases. Because mutations in MED17 result in autosomal recessive disorders, including microcephaly, intellectual disability, epilepsy, and ataxia, which are barely reported, with only three case reports to date, genotype–phenotype association should be elucidated. Here, we investigated the impact of MED17 mutations on cellular responses and found increased unfolded protein responses (UPRs) in fibroblasts derived from Japanese patients with MED17 mutations. The expression of the UPR genes CHOP and ATF4 was upregulated, and the phosphorylation of eIF2a was basally increased in patients’ cells. Based on our findings, we propose that increased UPRs caused by MED17 mutations might contribute to the clinical phenotype.
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Change history
13 September 2021
A Correction to this paper has been published: https://doi.org/10.1007/s10048-021-00669-y
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Acknowledgements
We thank the patients and their families for participation in this study. We also thank Shinichi Hirabayashi for clinical comments and cooperation of skin biopsy, and Edanz Group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript. S.H. was supported by grants from MEXT and JSPS (15K06758 and 19H04266).
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Fig. S1
Expression of mutated MED17 mRNA in the MED composition. a. Familial pedigree of two affected siblings with MED17 mutations and their healthy parents [11]. b. c.1013-5A>G splice site mutation produced irrelevant exon-exon junction, resulting in the translation of truncated proteins. c. Relative expression levels of MED17 in fibroblasts from MED17 patients. Cycloheximide (CHX, 50 μg/mL for 6 hrs) treatment increased the MED17 expression, especially using the primer containing the c.1013-5A>G mutation in patients' cells. Expression levels of each gene were normalized to those of GAPDH. Data are presented as means ± SD of three independent experiments. (PNG 155 kb)
Fig. S2
Expression of each MED subunits. a. MED17 antibody using in this study recognized both WT and c.1484T>G missense mutation of MED17 but not the truncated form. Immunoprecipitants from HeLa cells overexpressing a series of FLAG-tagged MED17 proteins were immunoblotted by FLAG antibody and MED17 antibody. b. Expression of MED proteins in fibroblasts from MED17 mutated patients and their healthy mothers were assessed by immunoblotting with the indicated antibodies. (PNG 162 kb)
Fig. S3
Irrelevant splicing of MED17 transcripts from c.1013-5A>G mutation allele. a. Splicing pattern of exon-exon junction of MED17 transcripts having c.1013-5A>G mutation. WT mRNA should be produced by accurate splicing, while four nucleotides would be inserted between exon 6 and 7 by irrelevant splicing. b. Experimental design of restriction fragment length polymorphism (RFLP) assay. cDNA reverse-transcribed from WT and c.1013-5A>G (accurate splicing) MED17 mRNA were digested to two DNA fragments by Mun I restriction enzyme, while c.1484T>G was not digested. DNA fragments from c.1013-5A>G (irrelevant splicing) was not determined without CHX because of the NMD. c. RFLP assay showed the absence of digested two DNA fragments in patients' cells indicating that predicted WT MED17 mRNA transcribed from c.1013-5A>G allele was barely expressed. (PNG 1658 kb)
Fig. S4
Expression of IEGs in fibroblasts from MED17 patients. Expression levels of each gene normalized to those of GAPDH are presented. Data are indicated as means ± SD of three independent experiments. There were no significant changes in the expression of each gene between patients and healthy mothers. (PNG 42 kb)
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Terabayashi, T., Hashimoto, S. Increased unfolded protein responses caused by MED17 mutations. Neurogenetics 22, 353–357 (2021). https://doi.org/10.1007/s10048-021-00661-6
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DOI: https://doi.org/10.1007/s10048-021-00661-6