An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing.
An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing.
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DOI：
10.1261/rna.2154310
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发表时间：
2010-06
期刊：
影响因子：
4.5
通讯作者：
N. Singh;K. Hollinger;Dhruva Bhattacharya;R. Singh
中科院分区：
文献类型：
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作者：
N. Singh;K. Hollinger;Dhruva Bhattacharya;R. Singh
在这里，我们报告了一个新的发现，反义寡核苷酸（阿索）微步，我们检查了位置特异性作用的内含子残基下游SMN 2外显子7的5'剪接位点（5' ss），跳跃与脊髓性肌萎缩症（SMA），婴儿死亡的主要遗传原因。我们的研究结果揭示了第10内含子位置（（10）C）的胞嘧啶残基的抑制作用，这是既不保守，也不与任何已知的剪接基序。在存在14聚体阿索（L14）的情况下，SMN 2外显子7的剪接模式出现了（10）C的显著性，该ASO（L14）隔离了（10）C下游的两个相邻hnRNP A1基序，但促进了SMN 2外显子7的跳跃。另一个14-mer阿索（F14）隔离了（10）C和相邻的hnRNP A1基序，导致SMN 2外显子7包含的强烈刺激。发现（10）C的抑制作用与其未配对状态和在靶向阿索与其内含子靶标之间形成的RNA：RNA螺旋的紧上游的特异性定位紧密相关。采用异源上下文以及改变的上下文SMN 2内含子7，我们表明，未配对的（10）C的抑制作用是依赖于一个长距离的相互作用，涉及下游内含子序列。我们的报告描述了一个罕见的例子，其中一个基于ASO的方法可以应用于解开内含子位置的关键作用，可能不属于线性基序，但通过长距离相互作用发挥重要作用。
Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5' splice site (5' ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position ((10)C), which is neither conserved nor associated with any known splicing motif. Significance of (10)C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from (10)C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, (10)C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of (10)C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired (10)C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions.