RNA structure drives interaction with proteins

ARTICLE https://doi.org/10.1038/s41467-019-10923-5 OPEN RNA structure drives interaction with proteins 1234567890():,; Natalia Sanchez de Groot 1,8, Alexandros Armaos1,8, Ricardo Graña-Montes1,7, Marion Alriquet2,3, Giulia Calloni2,3, R. Martin Vabulas2,3 & Gian Gaetano Tartaglia1,4,5,6 The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship —which we call structure-driven protein interactivity— allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs. 1 Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain. 2 Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany. 3 Institute of Biophysical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany. 4 ICREA 23 Passeig Lluis Companys 08010 and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain. 5 Department of Biology ‘Charles Darwin’, Sapienza University of Rome, P.le A. Moro 5, Rome 00185, Italy. 6 Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy. 7Present address: Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. 8These authors contributed equally: Natalia Sanchez de Groot, Alexandros Armaos. Correspondence and requests for materials should be addressed to R.M.V. (email: ) or to G.G.T. (email: ) NATURE COMMUNICATIONS | (2019)10:3246 | https://doi.org/10.1038/s41467-019-10923-5 | www.nature.com/naturecommunications 1 ARTICLE S NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10923-5 ince the central dogma was proposed in 1950, the main role attributed to RNA has been to act as the intermediate between DNA and protein synthesis. Yet, more than 70% of the genome is transcribed and just a small part codes for proteins1,2, which indicates that the majority of RNAs could have different biological roles. During the past decade many efforts were made to develop methods to study RNA isoforms: sequencing has been essential for detection of RNA species3 and recent developments provided a great deal of data on polymorphisms4, expression5 and half-lives6 of all types of RNAs, generating a valuable resource to understand their cellular functions and regulation. Although a number of techniques identified biological characteristics such as cellular location7 and secondary structure8,9, the characterization of the interaction network remains one of the most urgent challenges10,11. To this aim, computational methods are being developed to identify physicochemical features of the transcripts10, their conservation between species12 and, most importantly, binding partners13 that are also active in the cellular environment14. RNA is involved in many cellular processes such as control of gene expression, catalysis of various substrates, scaffolding of complex assemblies, and molecular chaperoning15. Its ability to act as a hub of cellular networks is at the centre of an active research field and has already led to the discovery of diverse ribonucleoprotein (RNP) assemblies16,17. A number of membrane-less organelles contain specific mixtures of RNAs and RBPs (RNA-binding proteins) that, due to their intrinsic lability, are difficult to characterize10. In most cases, liquid-like RNP assemblies, or condensates, such as P-bodies and stress granules18, exchange components with the surrounding content and adapt to the environmental condition in a dynamic way. Within these phase-separated assemblies RNA plays a central role19: whereas a polypeptide of 100 amino acids can interact with one or two proteins, a chain of 100 nucleotides is able to bind to 5–20 proteins, thus providing an ideal platform or scaffold for interactions20,21. Not surprisingly, changes in the interactions within RNP granules leading to liquid-to-solid phase transition are associated with the development of several human diseases, including neurological disorders and different types of cancer17. In RNP condensates such as stress granules, regulation of protein and RNA contacts is primarily controlled by HSP70 and cochaperones17 that act as versatile elements promoting assembly and disassembly of complexes22. In this large spectrum of activities, RNA structure controls the precise binding of proteins by creating spatial patterns and alternative conformations for the interactions to occur 12. Known complexes in which the structure regulates protein binding include transfer RNAs (tRNAs) whose three-dimensional conformation facilitates the codon/anticodon interaction23 and the ribosomal RNA (rRNA) scaffold that sustains the ribosome24. Importantly, the structure of a messenger RNA (mRNA) defines its lifecycle25, recruitment of ribosomes and response against environmental changes25. There are several cases of nucleotide chains of non-coding RNAs acting as scaffolds for protein complexes21: structured domains in NEAT1 attract paraspeckle components26 and repeat regions in XIST sequester proteins to orchestrate X-chromosome inactivation27. By contrast, poorly structured snoRNAs have been shown to facilitate the assembly of other transcripts28. Of both coding and non-coding transcripts, RBPs are known as the major regulators29 and are classified as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), depending on their binding preferences. Here we investigated the relationship between RNA structure and ability to interact with RBPs. At the transcriptome level, we find that the amount of RNA secondary structure correlates with the number of protein interactions. We 2 propose several possible implications of this relationship: a link to RNA types and biological roles; a connection to regulatory networks; and the ability to modulate phase separation. Based on our observations, we also demonstrated that this RNA property can be exploited in vitro to tune the contact network of a protein aggregate. Results Highly structured RNAs bind a large amount of proteins. With the aim of studying how RNA structure influences protein binding, we measured the amoun (...truncated)