Engreitz Jesse M, Sirokman Klara, McDonel Patrick, Shishkin Alexander A, Surka Christine, Russell Pamela, Grossman Sharon R, Chow Amy Y, Guttman Mitchell, Lander Eric S
Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Division of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA.
Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
Cell. 2014 Sep 25;159(1):188-199. doi: 10.1016/j.cell.2014.08.018.
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
许多非编码RNA（ncRNA）利用分子间RNA-RNA相互作用来实现其多样的功能。为了识别这些相互作用，我们开发了一种基于RNA反义纯化的方法来系统地绘制RNA-RNA相互作用图谱（RAP-RNA），并将其应用于研究与RNA加工相关的两种ncRNA：U1小核RNA（剪接体的一个组成部分）和Malat1（一种定位于核斑点的大型ncRNA）。U1和Malat1通过不同的靶向机制与新生转录本相互作用。利用差异交联，我们证实U1直接与整个内含子中的5'剪接位点和5'剪接位点基序杂交，并发现Malat1通过蛋白质中间体与前体mRNA间接相互作用。与新生前体mRNA的相互作用导致U1和Malat1在活跃基因处定位于靠近染色质的位置，这表明ncRNA可以利用RNA-RNA相互作用靶向特定的前体mRNA和基因组位点。RAP-RNA对丰度较低的RNA也很敏感，使其普遍适用于研究ncRNA。
