Molecular Basis of Two-Exon Skipping (Exons 12 and 13) by c.1248+5g>a in OXCT1 Gene: Study on Intermediates of OXCT1 Transcripts in Fibroblasts
Molecular Basis of Two-Exon Skipping (Exons 12 and 13) by c.1248+5g>a in OXCT1 Gene: Study on Intermediates of OXCT1 Transcripts in Fibroblasts
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DOI：
10.1002/humu.22258
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发表时间：
2013-03-01
期刊：
影响因子：
3.9
通讯作者：
Kondo, Naomi
中科院分区：
文献类型：
--
作者：
Hori, Tomohiro;Fukao, Toshiyuki;Kondo, Naomi
由剪接位点突变引起的同时两个外显子跳跃的分子基础尚未得到完全解释。 OXCT1 基因的剪接供体位点突变 c.1248+5g>a (IVS13) 主要导致琥珀酰辅酶 A:3-酮酸辅酶 A 转移酶 (SCOT) 缺陷患者 (GS23) 的成纤维细胞中外显子 12 和 13 的跳跃。我们比较了对照和 GS23 成纤维细胞之间的异核 RNA (hnRNA) 中间体。我们的策略是使用 hnRNA 的 RT-PCR 来检测包含连续外显子的 RNA 中间体 (SECRI) 中是否存在剪接外显子簇。我们最初的假设是，首先形成包含外显子 12 和 13 的 SECRI，然后在 GS23 细胞中跳过该 SECRI。然而，这样的途径被揭示并不是主要途径。因此，我们比较了对照和 GS23 之间 SCOT 转录本的内含子去除情况。在对照中，内含子 11 是最后一个被剪接的内含子，内含子 12 的去除也相当缓慢，并且发生在主要途径中内含子 13 去除之后。然而，GS23细胞中的突变导致内含子13保留，从而导致内含子12和11保留。这种剪接瘫痪可以通过跳过整个内含子11外显子12内含子12外显子13突变的内含子13，导致外显子12和13跳过来解决。
The molecular basis of simultaneous two-exon skipping induced by a splice-site mutation has yet to be completely explained. The splice donor site mutation c.1248+5g>a (IVS13) of the OXCT1 gene resulted predominantly in skipping of exons 12 and 13 in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared heteronuclear RNA (hnRNA) intermediates between controls' and GS23's fibroblasts. Our strategy was to use RT-PCR of hnRNA to detect the presence or absence of spliced exon clusters in RNA intermediates (SECRIs) comprising sequential exons. Our initial hypothesis was that a SECRI comprising exons 12 and 13 was formed first followed by skipping of this SECRI in GS23 cells. However, such a pathway was revealed to be not a major one. Hence, we compared the intron removal of SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced and the removal of intron 12 was also rather slow and occurred after the removal of intron 13 in a major pathway. However, the mutation in GS23 cells resulted in retention of intron 13, thus causing the retention of introns 12 and 11. This splicing paralysis may be solved by skipping the whole intron 11exon 12intron 12exon 13mutated intron 13, resulting in skipping of exons 12 and 13.