RNA-protein Interaction Networks Research Articles

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation invitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions

C9orf72 repeat expansions are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly(GR) proteins are toxic to neurons by forming cytoplasmic inclusions that sequester RNA-binding proteins including stress granule (SG) proteins. However, little is known of the factors governing poly(GR) inclusion formation. Here, we show that poly(GR) infiltrates a finely tuned network of protein-RNA interactions underpinning SG formation. It interacts with G3BP1, the key driver of SG assembly and a protein we found is critical for poly(GR) inclusion formation. Moreover, we discovered that N6-methyladenosine (m6A)-modified mRNAs and m6A-binding YTHDF proteins not only co-localize with poly(GR) inclusions in brains of c9FTD/ALS mouse models and patients with c9FTD, they promote poly(GR) inclusion formation via the incorporation of RNA into the inclusions. Our findings thus suggest that interrupting interactions between poly(GR) and G3BP1 or YTHDF1 proteins or decreasing poly(GR) altogether represent promising therapeutic strategies to combat c9FTD/ALS pathogenesis.

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN.

Heterotetrameric human transfer RNA (tRNA) splicing endonuclease TSEN catalyzes intron excision from precursor tRNAs (pre-tRNAs), utilizing two composite active sites. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH). Despite the essential function of TSEN, the three-dimensional assembly of TSEN-CLP1, the mechanism of substrate recognition, and the structural consequences of disease mutations are not understood in molecular detail. Here, we present single-particle cryogenic electron microscopy reconstructions of human TSEN with intron-containing pre-tRNAs. TSEN recognizes the body of pre-tRNAs and pre-positions the 3' splice site for cleavage by an intricate protein-RNA interaction network. TSEN subunits exhibit large unstructured regions flexibly tethering CLP1. Disease mutations localize far from the substrate-binding interface and destabilize TSEN. Our work delineates molecular principles of pre-tRNA recognition and cleavage by human TSEN and rationalizes mutations associated with PCH.

Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH.

G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

Single-Molecule Correlated Chemical Probing: A Revolution in RNA Structure Analysis.

RNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which involve treating an RNA with a reagent that modifies conformationally dynamic nucleotides, have broadly enabled examination of short- and long-range RNA structure in diverse contexts, including in living cells. For decades, chemical probing experiments have been interpreted in a per-nucleotide way, such that the reactivity measured at each nucleotide reports the average structure at a position over all RNA molecules within a sample. However, there are numerous important cases where per-nucleotide chemical probing falls short, including for RNAs that are bound by proteins, RNAs that form complex higher order structures, and RNAs that sample multiple conformations.Recent experimental and computational innovations have started a revolution in RNA structure analysis by transforming chemical probing into a massively parallel, single-molecule experiment. Enabled by a specialized reverse transcription strategy called mutational profiling (MaP), multiple chemical modification events can be measured within individual RNA molecules. Nucleotides that communicate structurally through direct base pairing or large-scale folding-unfolding transitions will react with chemical probes in a correlated manner, thereby revealing structural complexity hidden to conventional approaches. These single-molecule correlated chemical probing (smCCP) experiments can be interpreted to directly identify nucleotides that base pair (the PAIR-MaP strategy) and to reveal long-range, through-space structural communication (RING-MaP). Correlated probing can also define the thermodynamic populations of complex RNA ensembles (DANCE-MaP). Complex RNA-protein networks can be interrogated by cross-linking proteins to RNA and measuring correlations between cross-linked positions (RNP-MaP).smCCP thus visualizes RNA secondary and higher-order structure with unprecedented accuracy, defining novel structures, RNA-protein interaction networks, time-resolved dynamics, and allosteric structural switches. These strategies are not mutually exclusive; in favorable cases, multiple levels of RNA structure ─ base pairing, through-space structural communication, and equilibrium ensembles ─ can be resolved concurrently. The physical experimentation required for smCCP is profoundly simple, and experiments are readily performed in cells on RNAs of any size, including large noncoding RNAs and mRNAs. Single-molecule correlated chemical probing is paving the way for a new generation of biophysical studies on RNA in living systems.

Analysis of RNA-Protein Interaction Networks Using RNP-MaP.

RNA-protein interactions regulate a myriad of biological functions through formation of ribonucleoprotein complexes. These complexes may consist of one or more RNA-protein interaction network(s) providing additional layers of regulatory potential to the RNA. Moreover, since the protein-binding also regulates local and global structure of the RNA by structurally remodeling the latter, it is important to correlate RNA nucleotide flexibility with the site of protein-binding. We have discussed methods for chemical probing of structure of the RNA in the protein-free and protein-bound states in the preceding chapters. In this chapter, we describe a ribonucleoprotein mutational profiling (RNP-MaP) method for probing RNA-protein interaction networks.

Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP.

The functional roles of RNAs are often regulated by their structure. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) base reactivity can be employed to investigate the flexibility of nucleotides and correlate it to the constraints imparted by base-pairing and/or protein-binding. In vivo, a multitude of proteins could bind an RNA molecule, regulating its structure and function. Hence, to obtain a more comprehensive view of the RNA structure-function relationship in vivo, it may be required to characterize both the RNA structure and the RNA-protein interaction network. In this chapter, we describe methods for characterizing the in vivo nucleotide flexibility of RNA in cells by SHAPE-MaP (SHAPE by Mutational Profiling) and DMS-MaP. In another chapter, we will discuss the characterization of RNA-protein interaction network by RNP-MaP.

Cytoplasmic ribonucleoprotein complexes, RNA helicases and coronavirus infection

RNA metabolism in the eukaryotic cell includes the formation of ribonucleoprotein complexes (RNPs) that, depending on their protein components, have a different function. Cytoplasmic RNPs, such as stress granules (SGs) or P-bodies (PBs) are quite relevant during infections modulating viral and cellular RNA expression and as key players in the host cell antiviral response. RNA helicases are abundant components of RNPs and could have a significant effect on viral infection. This review focuses in the role that RNPs and RNA helicases have during coronavirus (CoVs) infection. CoVs are emerging highly pathogenic viruses with a large single-stranded RNA genome. During CoV infection, a complex network of RNA-protein interactions in different RNP structures is established. In general, RNA helicases and RNPs have an antiviral function, but there is limited knowledge on whether the viral protein interactions with cell components are mediators of this antiviral effect or are part of the CoV antiviral counteraction mechanism. Additional data is needed to elucidate the role of these RNA-protein interactions during CoV infection and their potential contribution to viral replication or pathogenesis.

De novo prediction of RNA-protein interactions with graph neural networks.

RNA-binding proteins (RBPs) are key co- and post-transcriptional regulators of gene expression, playing a crucial role in many biological processes. Experimental methods like CLIP-seq have enabled the identification of transcriptome-wide RNA-protein interactions for select proteins; however, the time- and resource-intensive nature of these technologies call for the development of computational methods to complement their predictions. Here, we leverage recent, large-scale CLIP-seq experiments to construct a de novo predictor of RNA-protein interactions based on graph neural networks (GNN). We show that the GNN method allows us not only to predict missing links in an RNA-protein network, but to predict the entire complement of targets of previously unassayed proteins, and even to reconstruct the entire network of RNA-protein interactions in different conditions based on minimal information. Our results demonstrate the potential of modern machine learning methods to extract useful information on post-transcriptional regulation from large data sets.

Identification and Validation of TRIM25 as a Glucose Metabolism Regulator in Prostate Cancer.

Prostate cancer (PCa) malignant progression is accompanied with the reprogramming of glucose metabolism. However, the genes involved in the regulation of glucose metabolism in PCa are not fully understood. Here, we propose a new method, DMRG, which constructs a weighted differential network (W-K-DN) to define the important metabolism-related genes. Based on biological knowledge and prostate cancer transcriptome data, a tripartite motif-containing 25 (TRIM25) was defined using DMRG; TRIM25 was involved in the regulation of glucose metabolism, which was verified by overexpressing or knocking down TRIM25 in PCa cell lines. Differential expression analysis of TCA cycle enzymes revealed that TRIM25 regulated isocitrate dehydrogenase 1 (IDH1) and fumarate hydratase (FH) expression. Moreover, a protein–RNA interaction network of TRIM25 revealed that TRIM25 interacted with RNA-binding proteins, including DExH-box helicase 9 and DEAD-box helicase 5, to play a role in regulating the RNA processing of metabolic enzymes, including IDH1 and FH. Furthermore, TRIM25 expression level was found to be positively correlated with Gleason scores in PCa patient tissues. In conclusion, this study provides a new method to define genes influencing tumor progression, and sheds light on the role of the defined TRIM25 in regulating glucose metabolism and promoting PCa malignancy.

RNA nucleoprotein complexes in biological systems

RNA-nucleoprotein (RNP) complexes are pivotal to the regulation of gene expression. These multiprotein, multifunctional complexes act as scaffolds, as molecular platforms, as hubs of interaction between the RNA and the proteins. The past decade has seen an exponential increase in the methodologies to identify and functionally characterize these complexes. In this review, we provide an overview of the recent advances in methodologies and the knowledge gained by characterizing these important complexes. This information has provided clues to the role of sequence, and structural determinants for RNA–Protein interaction networks in physiology and pathophysiology. With the recent studies implicating the role of RNA in the formation of biomolecular condensates, their role in health and disease warrants detailed sequence, structure and functional investigations.

Architecture of the cytoplasmic face of the nuclear pore.

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].

The Interplay Between Disordered Regions in RNAs and Proteins Modulates Interactions Within Stress Granules and Processing Bodies

Condensation, or liquid-like phase separation, is a phenomenon indispensable for the spatiotemporal regulation of molecules within the cell. Recent studies indicate that the composition and molecular organization of phase-separated organelles such as Stress Granules (SGs) and Processing Bodies (PBs) are highly variable and dynamic. A dense contact network involving both RNAs and proteins controls the formation of SGs and PBs and an intricate molecular architecture, at present poorly understood, guarantees that these assemblies sense and adapt to different stresses and environmental changes. Here, we investigated the physico-chemical properties of SGs and PBs components and studied the architecture of their interaction networks. We found that proteins and RNAs establishing the largest amount of contacts in SGs and PBs have distinct properties and intrinsic disorder is enriched in all protein-RNA, protein-protein and RNA-RNA interaction networks. The increase of disorder in proteins is accompanied by an enrichment in single-stranded regions of RNA binding partners. Our results suggest that SGs and PBs quickly assemble and disassemble through dynamic contacts modulated by unfolded domains of their components.

RNA-Protein Interaction Analysis of SARS-CoV-2 5' and 3' Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus. The viral genome is capped at the 5′ end, followed by an untranslated region (UTR). There is a poly(A) tail at the 3′ end, preceded by a UTR. The self-interaction between the RNA regulatory elements present within the 5′ and 3′ UTRs and their interaction with host/virus-encoded proteins mediate the function of the 5′ and 3′ UTRs. Using an RNA-protein interaction detection (RaPID) assay coupled to liquid chromatography with tandem mass spectrometry, we identified host interaction partners of SARS-CoV-2 5′ and 3′ UTRs and generated an RNA-protein interaction network. By combining these data with the previously known protein-protein interaction data proposed to be involved in virus replication, we generated the RNA-protein-protein interaction (RPPI) network, likely to be essential for controlling SARS-CoV-2 replication. Notably, bioinformatics analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism. Lysosome-associated membrane protein-2a (Lamp2a), the receptor for chaperone-mediated autophagy, is one of the host proteins that interact with the 5′ UTR. Further studies showed that the Lamp2 level is upregulated in SARS-CoV-2-infected cells and that the absence of the Lamp2a isoform enhanced the viral RNA level whereas its overexpression significantly reduced the viral RNA level. Lamp2a and viral RNA colocalize in the infected cells, and there is an increased autophagic flux in infected cells, although there is no change in the formation of autophagolysosomes. In summary, our study provides a useful resource of SARS-CoV-2 5′ and 3′ UTR binding proteins and reveals the role of Lamp2a protein during SARS-CoV-2 infection.IMPORTANCE Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins, and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5′ and 3′ UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication. Here, we identified the host proteins that associate with the UTRs of SARS-CoV-2, combined those data with the previously known protein-protein interaction data (expected to be involved in virus replication), and generated the RNA-protein-protein interaction (RPPI) network. Analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism, which are important for virus replication. Analysis of one of the interaction partners of the 5′-UTR (Lamp2a) demonstrated its role in reducing the viral RNA level in SARS-CoV-2-infected cells. Collectively, our study provides a resource of SARS-CoV-2 UTR-binding proteins and identifies an important role for host Lamp2a protein during viral infection.

Upregulation of long non-coding RNA SNHG16 promotes diabetes-related RMEC dysfunction via activating NF-κB and PI3K/AKT pathways

Diabetic retinopathy (DR) is a severe diabetes-induced eye disease, in which its pathological phenomena basically include abnormal proliferation, migration, and angiogenesis of microvascular endothelial cells in the retina. Long non-coding RNAs (lncRNAs) have been proven to be important regulators in various biological processes, but their participation in DR remains largely undiscovered. In the present study, we aimed to unveil the role of lncRNA small nucleolar RNA host gene 16 (SNHG16) in regulating the functions of human retinal microvascular endothelial cells (hRMECs) under a high-glucose (HG) condition. We found that SNHG16 expression was significantly upregulated in hRMECs treated with HG. Functionally, SNHG16 could facilitate hRMEC proliferation, migration, and angiogenesis. Moreover, SNHG16 was associated with nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Mechanistically, SNHG16 could promote hRMEC dysfunction by sequestering microRNA (miR)-146a-5p and miR-7-5p to act as a competing endogenous RNA (ceRNA) with interleukin-1 receptor-associated kinase 1 (IRAK1) and insulin receptor substrate 1 (IRS1). In conclusion, our results illustrated the potential role of SNHG16 in facilitating hRMEC dysfunction under HG treatment, providing a novel approach for DR therapy.

Advances in technologies for large-scale enrichment and identification of ribonucleic acid-protein complexes

核糖核酸(RNA)在细胞中并非单独存在,从它们产生到被降解的过程中与大量蛋白质发生相互作用,RNA结合蛋白(RNA-binding proteins, RBPs)能与RNA结合形成RNA-蛋白质复合物(RP复合物),并以这种复合物的形式发挥生理功能。RNAs或RBPs任一组分的异常与缺失都会影响RP复合物的正常生理功能,从而导致疾病的发生,如代谢异常、肌肉萎缩症、自身免疫性疾病和癌症。因此,定性定量分析RBPs及其在正常细胞和肿瘤细胞中与RNAs靶标之间的复杂相互作用网络有助于挖掘RP复合物在肿瘤发生发展中的作用,开发肿瘤生物标志物和新的治疗方式。要深入研究和理解RNAs与RBPs的相互作用网络,须依赖组学技术对RP复合物进行大规模鉴定。而作为在组学层面系统性解析RP复合物组成、含量和功能的第一步,大规模富集RP复合物极具挑战性。为了解决这一难题,研究者们发展了各种富集鉴定策略。该文针对RP复合物富集策略的最新进展进行了综述,包括紫外光交联和免疫沉淀(crosslinking and immunoprecipitation, CLIP)及其衍生技术、基于“点击化学”的富集策略和基于相分离的富集策略,比较分析了它们的技术原理、优缺点,以方便研究者们选择合适的策略来解决感兴趣的生物学问题。该文最后总结了当前的RP复合物富集方法仍然存在富集效率低和操作繁琐等亟需解决的技术挑战,为富集策略的发展提供了研究方向。

IdenPC-CAP: Identify protein complexes from weighted RNA-protein heterogeneous interaction networks using co-assemble partner relation.

Protein complexes play important roles in most cellular processes. The available genome-wide protein-protein interaction (PPI) data make it possible for computational methods identifying protein complexes from PPI networks. However, PPI datasets usually contain a large ratio of false positive noise. Moreover, different types of biomolecules in a living cell cooperate to form a union interaction network. Because previous computational methods focus only on PPIs ignoring other types of biomolecule interactions, their predicted protein complexes often contain many false positive proteins. In this study, we develop a novel computational method idenPC-CAP to identify protein complexes from the RNA-protein heterogeneous interaction network consisting of RNA-RNA interactions, RNA-protein interactions and PPIs. By considering interactions among proteins and RNAs, the new method reduces the ratio of false positive proteins in predicted protein complexes. The experimental results demonstrate that idenPC-CAP outperforms the other state-of-the-art methods in this field.

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library

Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale.

Analysis of RNA-protein networks with RNP-MaP defines functional hubs on RNA.

RNA-protein interaction networks govern many biological processes, but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a heterobifunctional crosslinker to freeze interacting proteins in place on RNA, and then maps multiple bound proteins in single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.

The role of 3'UTR-protein complexes in the regulation of protein multifunctionality and subcellular localization.

Multifunctional proteins often perform their different functions when localized in different subcellular compartments. However, the mechanisms leading to their localization are largely unknown. Recently, 3′UTRs were found to regulate the cellular localization of newly synthesized proteins through the formation of 3′UTR-protein complexes. Here, we investigate the formation of 3′UTR-protein complexes involving multifunctional proteins by exploiting large-scale protein-protein and protein-RNA interaction networks. Focusing on 238 human ‘extreme multifunctional’ (EMF) proteins, we predicted 1411 3′UTR-protein complexes involving 54% of those proteins and evaluated their role in regulating protein cellular localization and multifunctionality. We find that EMF proteins lacking localization addressing signals, yet present at both the nucleus and cell surface, often form 3′UTR-protein complexes, and that the formation of these complexes could provide EMF proteins with the diversity of interaction partners necessary to their multifunctionality. Our findings are reinforced by archetypal moonlighting proteins predicted to form 3′UTR-protein complexes. Finally, the formation of 3′UTR-protein complexes that involves up to 17% of the proteins in the human protein-protein interaction network, may be a common and yet underestimated protein trafficking mechanism, particularly suited to regulate the localization of multifunctional proteins.