Short intronic repeat sequences facilitate circular RNA production.
Short intronic repeat sequences facilitate circular RNA production.
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DOI：
10.1101/gad.251926.114
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发表时间：
2014-10-15
影响因子：
10.5
通讯作者：
Wilusz JE
中科院分区：
文献类型：
--
作者：
Liang D;Wilusz JE
最近的深度测序研究揭示了数千个由蛋白质编码基因产生的环状非编码RNA。剪接体仅选择某些外显子进行循环的机制在很大程度上尚不清楚。梁和威卢兹使用了广泛的表达质粒突变来证明，包含剪接位点和短(∼30-到40-nT)反向重复的微型内含子足以使插入的外显子在细胞中循环。内含子重复序列和外显子序列必须相互协作，并且需要一个功能性的3‘端处理信号，这表明环化可能发生在转录后。最近的深度测序研究揭示了数千个由蛋白质编码基因产生的环状非编码RNA。这些RNA是当前体信使RNA(前mRNA)剪接机制“反向剪接”并共价连接在一起时产生的，例如，单个外显子的两端。然而，剪接体仅选择某些外显子进行循环的机制在很大程度上尚不清楚。通过对表达质粒的广泛突变，我们证明了包含剪接位点和短(∼30-40-核苷酸)反向重复的微型内含子，如Alu元件，足以使插入的外显子在细胞中循环。内含子重复序列必须彼此碱基配对，从而使剪接位点彼此接近。然而，显然不止是简单的热力学在起作用，因为并不是所有的重复都支持环化，而增加重复之间的发夹的稳定性有时会抑制循环RNA的生物发生。内含子重复序列和外显子序列必须相互协作，并且需要一个功能性的3‘端处理信号，这表明环化可能发生在转录后。这些结果提出了详细和可推广的模型，解释了剪接机制如何决定是产生环形非编码RNA还是产生线性RNA。
Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. The mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Liang and Wilusz used extensive mutagenesis of expression plasmids to show that miniature introns containing the splice sites along with short (∼30- to 40-nt) inverted repeats are sufficient to allow the intervening exons to circularize in cells. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.
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