Higher-Order Organization Principles of Pre-translational mRNPs

• Published in: Molecular cell Vol. 72; no. 4; pp. 715 - 726.e3
• Main Authors: Metkar, Mihir, Ozadam, Hakan, Lajoie, Bryan R., Imakaev, Maxim, Mirny, Leonid A., Dekker, Job, Moore, Melissa J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.11.2018
• Subjects: Cell Nucleus; ChimeraTie; exon-junction complex; Exons; HEK293 Cells; Humans; Immunoprecipitation - methods; mRNA; mRNP structure; Protein Processing, Post-Translational; proximity ligation; Ribonucleoproteins - genetics; Ribonucleoproteins - ultrastructure; RIPPLiT; RNA Precursors - genetics; RNA Precursors - ultrastructure; RNA Splicing; RNA Stability; RNA, Messenger - genetics; RNA, Messenger - ultrastructure; RNA, Untranslated; Spliceosomes; Transcription, Genetic; mRNP structure; exon-junction complex; ChimeraTie; mRNA; RIPPLiT; proximity ligation
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.09.012

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. [Display omitted] •RIPPLiT captures 3D structure information in diverse RNA protein complexes (RNPs)•RIPPLiT provides RNA-RNA proximity information independent of secondary structure•Spliced mRNAs behave like flexible polymers without strong locus-specific structures•Polymer analysis reveals that pre-translational mRNPs form linear rods Metkar et al. developed a new toolkit, a biochemical approach, RIPPLiT and a bioinformatics suite to capture and analyze higher-order organization of RNPs. They were able to enrich for interactions within mRNAs and further, using polymer analysis on 100 s of mRNAs, identified a unifying principle for mRNP packaging.