Jones Alisha N, Tikhaia Ekaterina, Mourão André, Sattler Michael
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85747, Garching, Germany.
Nucleic Acids Res. 2022 Feb 28;50(4):2350-2362. doi: 10.1093/nar/gkac080.
The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5' stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure.
lncRNA Xist的A重复区域对于X染色体失活至关重要，并含有多个N6-甲基腺嘌呤（m6A）修饰。m6A修饰如何影响A重复序列保守的AUCG四环发夹的构象以及它如何被YTHDC1阅读蛋白识别尚不清楚。在此，我们报道了（m6A）UCG发夹的核磁共振溶液结构，该结构揭示m6A碱基延伸了A-form螺旋茎的5'堆积，类似于未甲基化的AUCG四环。YTHDC1与（m6A）UCG四环结合的晶体结构表明，（m6A）UC核苷酸以单链构象被YTHDC1的YTH结构域识别。m6A碱基插入芳香笼中，U和C碱基与侧翼带电荷的表面区域相互作用，类似于对单链m6A RNA配体的识别。值得注意的是，核磁共振和荧光猝灭实验表明，结合需要（m6A）UCG发夹上部茎区域的局部解折叠。我们的数据表明，m6A可以很容易地容纳在发夹环区域，但YTH阅读蛋白的识别需要侧翼茎区域的局部解折叠。这表明m6A修饰可能通过调节RNA结构来调控lncRNA功能。
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