Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK.
London Neurodegenerative Disease Brain Bank, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 8AF, UK.
Acta Neuropathol Commun. 2016 Feb 25;4:18. doi: 10.1186/s40478-016-0289-4.
The most common forms of amyotrophic lateral sclerosis and frontotemporal dementia are caused by a large GGGGCC repeat expansion in the first intron of the C9orf72 gene. The repeat-containing intron should be degraded after being spliced out, however GGGGCC repeat-containing RNA species either accumulate in nuclear foci or are exported to the cytoplasm where they are translated into potentially toxic dipeptide repeat proteins by repeat-associated non-AUG-initiated (RAN) translation.
In order to determine the mechanisms of repeat-containing intron misprocessing, we have analyzed C9orf72 transcripts in lymphoblasts from C9orf72 expansion carriers (n = 15) and control individuals (n = 15). We have identified polyadenylated C9orf72 RNA species retaining the repeat-containing intron and in which downstream exons are spliced correctly resulting in a C9orf72 mRNA with an enlarged 5'-UTR containing the GGGGCC repeats. Intron-retaining transcripts are produced from both wild-type and mutant alleles. Intron-retaining C9orf72 transcripts were also detected in brain with a 2.7 fold increase measured in the frontal cortex from heterozygous expansion carriers (n = 11) compared to controls (n = 10). The level of intron-retaining transcripts was increased 5.9 fold in a case homozygous for the expansion. We also show that a large proportion of intron 1-retaining C9orf72 transcripts accumulate in the nucleus.
Retention of the repeat-containing intron in mature C9orf72 mRNA can potentially explain nuclear foci formation as well as nuclear export of GGGGCC repeat RNA and suggests that the misprocessing of C9orf72 transcripts initiates the pathogenic process caused by C9orf72 hexanucleotide repeat expansions as well as provides the basis for novel therapeutic strategies.
最常见的肌萎缩性侧索硬化症和额颞叶痴呆是由 C9orf72 基因第一内含子中的大 GGGGCC 重复扩展引起的。然而，带有重复的内含子在被剪接后应该被降解，但是含有 GGGGCC 重复的 RNA 物种要么在核斑点中积累，要么被输出到细胞质中，在那里它们通过重复相关的非 AUG 起始（RAN）翻译被翻译成潜在毒性的二肽重复蛋白。
为了确定重复内含子错误加工的机制，我们分析了 C9orf72 扩张携带者（n=15）和对照个体（n=15）的淋巴母细胞中的 C9orf72 转录本。我们鉴定了含有重复的内含子并正确剪接下游外显子的多聚腺苷酸化 C9orf72 RNA 物种，从而产生具有包含 GGGGCC 重复的扩大 5'-UTR 的 C9orf72 mRNA。内含子保留转录本是由野生型和突变型等位基因产生的。在杂合性扩张携带者的额皮质中也检测到了含有内含子的 C9orf72 转录本，与对照组相比，（n=11）增加了 2.7 倍（n=10）。在一个扩张的纯合子病例中，内含子保留转录本的水平增加了 5.9 倍。我们还表明，大量的内含子 1 保留的 C9orf72 转录本在核内积累。
成熟的 C9orf72 mRNA 中重复内含子的保留可能可以解释核斑点的形成以及 GGGGCC 重复 RNA 的核输出，并表明 C9orf72 转录本的错误加工引发了由 C9orf72 六核苷酸重复扩展引起的致病过程，并为新的治疗策略提供了基础。
