ABSTRACT

lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNA structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their downstream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs, we demonstrate first the significant association of an SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.

摘要

lncRNAs是许多调控过程的核心，也被认为与各种复杂疾病有关。它们通过与 RNA、DNA 或蛋白质的直接相互作用来影响基因调控。因此，lncRNA 结构可能对其调节功能至关重要。点突变，在基因组筛选中表现为 SNP（单核苷酸多态性），可以显着改变它们的功能，并随后改变它们下游调节基因的表达。为了测试 SNP 对结构的影响，我们研究了与扩张型心肌病相关的 lncRNA。在 322 名人类候选 lncRNA 中，我们首先使用来自 1084 名患病和 751 名对照患者的数据证明了位于 lncRNA H19 中的 SNP 的显着关联。H19通常在心脏中高度表达，在心脏发育过程中具有复杂的表达模式。接下来，我们使用 MFE（最小自由能）折叠来证明这个 861 nt 长的 lncRNA 的二级结构发生了显着的重新折叠。由于 MFE 折叠可能会忽略次优结构的重要性，我们表明这种重新折叠也体现在整个玻尔兹曼结构集合中。在那里，结构的组成在其热力学概率方面受到遗传变异的极大影响。最后，我们使用 SHAPE-Seq 通过实验证实了这些结果，证实了影响这些结构的 SNP 可以解释隐藏的遗传变异，而这些变异并未通过全基因组关联研究得到解释。我们的研究结果表明 lncRNA 的结构变化，尤其是 lncRNA H19，