G-quadruplex formation enhances splicing efficiency of PAX9 intron 1
G-quadruplex formation enhances splicing efficiency of PAX9 intron 1
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DOI：
10.1007/s00439-014-1485-6
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发表时间：
2015-01-01
期刊：
影响因子：
5.3
通讯作者：
Peres Line, Sergio Roberto
中科院分区：
文献类型：
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作者：
Ribeiro, Mariana Martins;Teixeira, Gleidson Silva;Peres Line, Sergio Roberto
G-四链体是存在于DNA和RNA分子中的二级结构，其通过G-四联体（即，通过Hoogsteen氢键结合的四个鸟嘌呤（G-束）的相互作用）。人PAX 9内含子1具有位于外显子1附近的推定的G-四链体形成区，其存在于所有已知测序的胎盘哺乳动物中。使用圆二色性（CD）分析和CD熔解，我们表明，这些序列能够形成高度稳定的四链体结构。由于四链体结构接近外显子-内含子边界，我们使用经验证的双报告剪接测定和qPCR来分析其对剪接效率的作用。人四链体显示出对PAX 9内含子1的剪接效率具有关键作用，因为消除四链体形成的突变显著降低了人PAX 9内含子1的剪接效率。与人类序列相比，不太稳定的大鼠四链体的剪接效率较低。此外，用360 A（一种稳定四链体结构的强配体）处理进一步增加了人PAX 9内含子1的剪接效率。总之，这些结果提供了证据，G-四链体结构参与PAX 9内含子1的剪接效率。
G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.