Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles.
Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles.
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DOI：
10.1261/rna.059477.116
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发表时间：
2017-06
期刊：
影响因子：
--
通讯作者：
Fox AH
中科院分区：
文献类型：
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作者：
Li R;Harvey AR;Hodgetts SI;Fox AH
近年来发现了大量的长非编码RNA，但只有少数得到了表征。 NEAT1（核副斑点组装转录本 1）是一种哺乳动物长非编码 RNA，对于小鼠的生殖生理学、癌症发展以及称为副斑点的亚核体的形成非常重要。 NEAT1 的两种主要亚型（人体内为 3.7 kb NEAT1_1 和 23 kb NEAT1_2）由共同启动子产生，并通过使用替代转录终止位点产生。这种基因结构使得两种亚型之间的功能关系难以剖析。在这里，我们使用 CRISPR-Cas9 基因组编辑创建了几种不同的细胞系：完全 NEAT1 敲除细胞、仅表达短形式 NEAT1_1 的细胞以及具有两倍 NEAT1_2 的细胞。使用这些试剂，我们获得了 NEAT1_1 不是副斑雀的主要成分的证据。此外，我们的数据表明 NEAT1_1 定位于许多我们称为“微散斑”的非副散斑焦点，这些焦点可能具有与副散斑无关的功能。这项研究强调了 lncRNA 的复杂性，并展示了基因组编辑工具如何在剖析重叠转录本的结构和功能作用方面发挥作用。
Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed “microspeckles,” which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.