Haplotype-specific MAPT exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders.
Haplotype-specific MAPT exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders.
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DOI：
10.1186/s13024-017-0224-6
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发表时间：
2017-10-30
影响因子：
15.1
通讯作者：
Caffrey TM
中科院分区：
文献类型：
--
作者：
Lai MC;Bechy AL;Denk F;Collins E;Gavriliouk M;Zaugg JB;Ryan BJ;Wade-Martins R;Caffrey TM
全基因组关联研究已经将微管相关蛋白tau（MAPT）H1单倍型单核苷酸多态性（SNP）鉴定为帕金森病、进行性核上性麻痹和皮质基底节变性的主要常见风险变体。MAPT风险变异体落入高连锁不平衡的大的1.8Mb区域内，使得难以辨别功能上重要的风险变异体。在这里，我们利用MAPT外显子3的强单倍型特异性表达来研究位于连锁不平衡的H1单倍型区域内的SNP的功能。在这项研究中，我们剖析的分子机制，单倍型特异性SNP赋予等位基因特异性的MAPT外显子3的选择性剪接的影响。首先，我们使用单倍型杂交全基因座基因组MAPT载体研究来鉴定功能性SNPs。接下来，我们通过质谱分析两个位点的RNA-蛋白质相互作用。最后，我们敲低候选剪接因子，以确定其对MAPT外显子3的影响，使用一种新的等位基因特异性qPCR测定。使用全基因座基因组DNA表达载体表达MAPT单倍型变体，我们证明，rs 17651213调节外显子3包含在单倍型特异性的方式。我们进一步研究了该区域的功能，使用RNA电泳迁移率变动分析，以显示差异RNA-蛋白质复合物的形成在H1和H2序列的SNP rs 17651213和rs 1800547的变体，随后确定候选人的反式作用剪接因子与这些功能性SNP序列的RNA-蛋白质下拉实验和质谱。最后，通过质谱鉴定的候选剪接因子的基因敲除证明了hnRNP F和hnRNP Q在外显子3包含的单倍型特异性调节中的作用。我们确定了常见的剪接因子hnRNP F和hnRNP Q调节单倍型特异性剪接的MAPT外显子3通过内含子变体rs 1800547和rs 17651213。这项工作展示了一个综合的方法来验证大区域的连锁不平衡的风险变异的功能。本文的在线版本（10.1186/s13024-017-0224-6）包含补充材料，可供授权用户使用。
Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium. In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay. Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion. We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium. The online version of this article (10.1186/s13024-017-0224-6) contains supplementary material, which is available to authorized users.
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影响因子：
14.9
作者：
Desmet FO;Hamroun D;Lalande M;Collod-Béroud G;Claustres M;Béroud C
通讯作者：
Béroud C
影响因子：
6.1
作者：
Caffrey, Tara M.;Wade-Martins, Richard
通讯作者：
Wade-Martins, Richard
影响因子：
14.9
作者：
ANDREADIS, A;BRODERICK, JA;KOSIK, KS
通讯作者：
KOSIK, KS
影响因子：
3.5
作者：
Baker, M;Litvan, I;Hutton, M
通讯作者：
Hutton, M
影响因子：
4.4
作者：
Hecht M;Bromberg Y;Rost B
通讯作者：
Rost B