SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein–RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure–function interrelationships for Xist and other lncRNAs in cells.

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RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

10.1101/2020.02.07.939108 ◽

2020 ◽

Cited By ~ 2

Author(s):

Chase A. Weidmann ◽

Anthony M. Mustoe ◽

Parth B. Jariwala ◽

J. Mauro Calabrese ◽

Kevin M. Weeks

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

Noncoding Rna ◽

Interaction Network ◽

Protein Interaction Networks ◽

Interaction Networks ◽

E Region ◽

Nucleotide Resolution ◽

Map Data ◽

In Cells

ABSTRACTRNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but these networks are currently impossible to examine in a comprehensive way. We developed a live-cell chemical probing strategy for mapping protein interaction networks in any RNA with single-nucleotide resolution. This RNP-MaP strategy (RNP network analysis by mutational profiling) simultaneously detects binding by and cooperative interactions involving multiple proteins with single RNA molecules. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein interaction network hubs identify function-critical sites in these RNAs. RNP-MaP identified numerous protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and distinguished communities of proteins that network together on XIST. RNP-MaP data show that the Xist E region is densely networked by protein interactions and that PTBP1, MATR3, and TIA1 proteins each interface with the XIST E region via two distinct interaction modes; and we find that the XIST E region is sufficient to mediate RNA foci formation in cells. RNP-MaP will enable discovery and mechanistic analysis of protein interaction networks across any RNA in cells.

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POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905381116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23527-23533 ◽

Cited By ~ 9

Author(s):

Mengyuan Xu ◽

Janna Kiselar ◽

Tawna L. Whited ◽

Wilnelly Hernandez-Sanchez ◽

Derek J. Taylor

Keyword(s):

Conformational Changes ◽

Protein Interactions ◽

Environmental Changes ◽

Lymphocytic Leukemia ◽

Protein Protein Interactions ◽

Cellular Environment ◽

Multiple Protein ◽

Linear Chromosomes ◽

Hydroxyl Radical Footprinting ◽

Regulate Telomere Length

Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein–protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.

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Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells

RNA ◽

10.1261/rna.078896.121 ◽

2021 ◽

pp. rna.078896.121

Author(s):

Yan Han ◽

Xuzhen Guo ◽

Tiancai Zhang ◽

Jiangyun Wang ◽

Keqiong Ye

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

Rna Binding ◽

Rna Binding Proteins ◽

High Specificity ◽

Ester Group ◽

Yeast Cells ◽

Primary Amines ◽

Low Efficiency ◽

In Cells

Characterization of RNA-protein interaction is fundamental for understanding metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

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MOHCA-seq: RNA 3D models from single multiplexed proximity-mapping experiments

10.1101/004556 ◽

2014 ◽

Cited By ~ 3

Author(s):

Clarence Cheng ◽

Fang-Chieh Chou ◽

Wipapat Kladwang ◽

Siqi Tian ◽

Pablo Cordero ◽

...

Keyword(s):

Conformational Changes ◽

Deep Sequencing ◽

Structure Determination ◽

Rna Structure ◽

Tertiary Structure ◽

3D Models ◽

Biological Processes ◽

3D Information ◽

Nucleotide Resolution

Large RNAs control myriad biological processes but challenge tertiary structure determination. We report that integrating Multiplexed OH Cleavage Analysis with tabletop deep sequencing (MOHCA-seq) gives nucleotide-resolution proximity maps of RNA structure from single straightforward experiments. After achieving 1-nm resolution models for RNAs of known structure, MOHCA-seq reveals previously unattainable 3D information for ligand-induced conformational changes in a double glycine riboswitch and the sixth community-wide RNA puzzle, an adenosylcobalamin riboswitch.

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RNA structure probing to characterize RNA-protein interactions on a low abundance pre-mRNA in living cells

RNA ◽

10.1261/rna.077263.120 ◽

2020 ◽

pp. rna.077263.120

Author(s):

Jodi L Bubenik ◽

Melissa Hale ◽

Ona McConnell ◽

Eric Wang ◽

Maurice S. Swanson ◽

...

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

Living Cells ◽

Structure Probing

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Applications of In-Cell NMR in Structural Biology and Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms20010139 ◽

2019 ◽

Vol 20 (1) ◽

pp. 139 ◽

Cited By ~ 9

Author(s):

CongBao Kang

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Protein Interactions ◽

Mammalian Cells ◽

Structural Information ◽

Living Cells ◽

Bacterial Cells ◽

Protein Protein Interactions ◽

Nmr Studies ◽

Post Translational Modifications

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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Capturing RNA–protein interaction via CRUIS

Nucleic Acids Research ◽

10.1093/nar/gkaa143 ◽

2020 ◽

Vol 48 (9) ◽

pp. e52-e52 ◽

Cited By ~ 5

Author(s):

Ziheng Zhang ◽

Weiping Sun ◽

Tiezhu Shi ◽

Pengfei Lu ◽

Min Zhuang ◽

...

Keyword(s):

Mass Spectrometry ◽

Dna Damage ◽

Protein Interactions ◽

Noncoding Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Living Cells ◽

Innovative Methods ◽

Rna Biology

Abstract No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology.

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Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

10.1101/488163 ◽

2018 ◽

Author(s):

Sindhuja Sridharan ◽

Nils Kurzawa ◽

Thilo Werner ◽

Ina Guenthner ◽

Dominic Helm ◽

...

Keyword(s):

Thermal Stability ◽

Protein Interactions ◽

Protein Complexes ◽

Nucleic Acid Binding ◽

Macromolecular Assemblies ◽

Intrinsically Disordered ◽

Cellular Environment ◽

Nucleic Acid Binding Proteins ◽

High Concentrations ◽

In Cells

Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules. Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes and membrane-less organelles. Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates. However, system-wide studies to characterize NTP- interactions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 μM, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

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The intracellular environment affects protein–protein interactions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019918118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2019918118

Author(s):

Shannon L. Speer ◽

Wenwen Zheng ◽

Xin Jiang ◽

I-Te Chu ◽

Alex J. Guseman ◽

...

Keyword(s):

Protein Interactions ◽

Protein Complex ◽

Hard Core ◽

Protein Protein Interactions ◽

Chemical Interactions ◽

E Coli ◽

Cellular Environment ◽

High Concentrations ◽

In Cells

Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.

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A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802429115 ◽

2018 ◽

Vol 115 (25) ◽

pp. 6404-6409 ◽

Cited By ~ 31

Author(s):

Anna-Lena Steckelberg ◽

Benjamin M. Akiyama ◽

David A. Costantino ◽

Tim L. Sit ◽

Jay C. Nix ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Rna Structure ◽

Noncoding Rna ◽

Sequence Similarity ◽

Functional Class ◽

Biologically Active ◽

X Ray Crystallography ◽

Single Molecule Fret ◽

Rna Maturation

Folded RNA elements that block processive 5′ → 3′ cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

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