RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Mol Cell. 2018 Nov 15;72(4):715-726.e3. doi: 10.1016/j.molcel.2018.09.012. Epub 2018 Nov 8.
Compared to noncoding RNAs (ncRNAs), such as rRNAs and ribozymes, for which high-resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNA particles (mRNPs), but the manner of this mRNA compaction is unknown. Here, we developed and implemented RIPPLiT (RNA immunoprecipitation and proximity ligation in tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably associated with defined proteins, in this case, exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton-sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNPs, wherein strong locus-specific structures predominate, mRNPs behave more like flexible polymers. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that nascent and pre-translational mammalian mRNAs are compacted by their associated proteins into linear rod-like structures.
与富含高分辨率结构的非编码 RNA（ncRNA），如 rRNA 和核酶相比，信使 RNA（mRNA）的三级结构知之甚少。在真核细胞中，新合成的 mRNA 与蛋白质在高度浓缩的 mRNA 颗粒（mRNP）中被包装，但这种 mRNA 浓缩的方式尚不清楚。在这里，我们开发并实施了 RIPPLiT（串联 RNA 免疫沉淀和邻近连接），这是一种在全转录组范围内探测与特定蛋白质稳定相关的 RNA 三维构象的方法，在这种情况下，是外显子结合复合物（EJC）核心因子。EJCs 与其他 mRNP 成分多聚化形成兆道尔顿大小的复合物，保护大量新合成的 mRNA 免受内切酶的消化。与 ncRNP 不同，ncRNP 中主要存在强烈的基因座特异性结构，mRNP 更像是柔性聚合物。对数百种 mRNA 种属邻近连接数据的聚合物分析表明，新生和翻译前的哺乳动物 mRNA 与其相关蛋白一起被包装成线性棒状结构。
