scholarly journals Dynamic Regulation of RNA Structure in Mammalian Cells

2018 ◽  
Author(s):  
Lei Sun ◽  
Furqan Fazal ◽  
Pan Li ◽  
James P. Broughton ◽  
Byron Lee ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Structural Information ◽  
Rna Structures ◽  
Rna Modifications ◽  
Dynamic Regulation ◽  
Comparative Structure

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, termed RNA structuromes. However, previous whole-cell analyses lacked the resolution to unravel the dynamic regulation of RNA structure across subcellular states. Here we reveal the RNA structuromes in three compartments — chromatin, nucleoplasm and cytoplasm. The cytotopic structuromes substantially expand RNA structural information, and enable detailed investigation of the central role of RNA structure in linking transcription, translation, and RNA decay. Through comparative structure analysis, we develop a resource to visualize the interplay of RNA-protein interactions, RNA chemical modifications, and RNA structure, and predict both direct and indirect reader proteins of RNA modifications. We validate the novel role of the RNA binding protein LIN28A as an N6-methyladenosine (m6A) modification “anti-reader”. Our results highlight the dynamic nature of RNA structures and its functional significance in gene regulation.

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

RNA ◽  
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.

scholarly journals Protein-assisted RNA fragment docking (RnaX) for modeling RNA–protein interactions using ModelX

2019 ◽  
Vol 116 (49) ◽  
pp. 24568-24573 ◽  
Author(s):  
Javier Delgado Blanco ◽  
Leandro G. Radusky ◽  
Damiano Cianferoni ◽  
Luis Serrano
Keyword(s):  
Conformational Changes ◽  
Protein Design ◽  
Protein Interactions ◽  
Rna Binding ◽  
Structural Information ◽  
Conformational Dynamics ◽  
Computational Cost ◽  
Regulation Of Transcription ◽  
Sequence Specificity ◽  
Protein Interfaces

RNA–protein interactions are crucial for such key biological processes as regulation of transcription, splicing, translation, and gene silencing, among many others. Knowing where an RNA molecule interacts with a target protein and/or engineering an RNA molecule to specifically bind to a protein could allow for rational interference with these cellular processes and the design of novel therapies. Here we present a robust RNA–protein fragment pair-based method, termed RnaX, to predict RNA-binding sites. This methodology, which is integrated into the ModelX tool suite (http://modelx.crg.es), takes advantage of the structural information present in all released RNA–protein complexes. This information is used to create an exhaustive database for docking and a statistical forcefield for fast discrimination of true backbone-compatible interactions. RnaX, together with the protein design forcefield FoldX, enables us to predict RNA–protein interfaces and, when sufficient crystallographic information is available, to reengineer the interface at the sequence-specificity level by mimicking those conformational changes that occur on protein and RNA mutagenesis. These results, obtained at just a fraction of the computational cost of methods that simulate conformational dynamics, open up perspectives for the engineering of RNA–protein interfaces.

scholarly journals Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control

2020 ◽  
Vol 21 (14) ◽  
pp. 5161 ◽  
Author(s):  
Katarzyna Taylor ◽  
Krzysztof Sobczak
Keyword(s):  
Alternative Splicing ◽  
Rna Structure ◽  
The Other ◽  
Splicing Regulation ◽  
Rna Structures ◽  
Structural Arrangement ◽  
Biological Functionality ◽  
Cis And Trans ◽  
Structural Aspects

Alternative splicing is a highly sophisticated process, playing a significant role in posttranscriptional gene expression and underlying the diversity and complexity of organisms. Its regulation is multilayered, including an intrinsic role of RNA structural arrangement which undergoes time- and tissue-specific alterations. In this review, we describe the principles of RNA structural arrangement and briefly decipher its cis- and trans-acting cellular modulators which serve as crucial determinants of biological functionality of the RNA structure. Subsequently, we engage in a discussion about the RNA structure-mediated mechanisms of alternative splicing regulation. On one hand, the impairment of formation of optimal RNA structures may have critical consequences for the splicing outcome and further contribute to understanding the pathomechanism of severe disorders. On the other hand, the structural aspects of RNA became significant features taken into consideration in the endeavor of finding potential therapeutic treatments. Both aspects have been addressed by us emphasizing the importance of ongoing studies in both fields.

scholarly journals Methionine adenosyltransferases in cancers: Mechanisms of dysregulation and implications for therapy

2017 ◽  
Vol 243 (2) ◽  
pp. 107-117 ◽  
Author(s):  
Lauren Y Maldonado ◽  
Diana Arsene ◽  
José M Mato ◽  
Shelly C Lu
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Methionine Adenosyltransferase ◽  
Therapeutic Interventions ◽  
Cancer Development ◽  
Growth And Survival ◽  
Liver Cancers ◽  
Mat Genes

Methionine adenosyltransferase genes encode enzymes responsible for the biosynthesis of S-adenosylmethionine, the principal biological methyl donor and precursor of polyamines and glutathione. Mammalian cells express three genes – MAT1A, MAT2A, and MAT2B – with distinct expression and functions. MAT1A is mainly expressed in the liver and maintains the differentiated states of both hepatocytes and bile duct epithelial cells. Conversely, MAT2A and MAT2B are widely distributed in non-parenchymal cells of the liver and extrahepatic tissues. Increasing evidence suggests that methionine adenosyltransferases play significant roles in the development of cancers. Liver cancers, namely hepatocellular carcinoma and cholangiocarcinoma, involve dysregulation of all three methionine adenosyltransferase genes. MAT1A reduction is associated with increased oxidative stress, progenitor cell expansion, genomic instability, and other mechanisms implicated in tumorigenesis. MAT2A/MAT2B induction confers growth and survival advantage to cancerous cells, enhancing tumor migration. Highlighted examples from colon, gastric, breast, pancreas and prostate cancer studies further underscore methionine adenosyltransferase genes’ role beyond the liver in cancer development. In this subset of extra-hepatic cancers, MAT2A and MAT2B are induced via different regulatory mechanisms. Understanding the role of methionine adenosyltransferase genes in tumorigenesis helps identify attributes of these genes that may serve as valuable targets for therapy. While S-adenosylmethionine, and its metabolite, methylthioadenosine, have been largely explored as therapeutic interventions, targets aimed at regulation of MAT gene expression and methionine adenosyltransferase protein–protein interactions are now surfacing as potential effective strategies for treatment and chemoprevention of cancers. Impact statement This review examines the role of methionine adenosyltransferases (MATs) in human cancer development, with a particular focus on liver cancers in which all three MAT genes are implicated in tumorigenesis. An overview of MAT genes, isoenzymes and their regulation provide context for understanding consequences of dysregulation. Highlighting examples from liver, colon, gastric, breast, pancreas and prostate cancers underscore the importance of understanding MAT’s tumorigenic role in identifying future targets for cancer therapy.

scholarly journals Shaping the regulation of the p53 mRNA tumour suppressor: the co-evolution of genetic signatures

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Konstantinos Karakostis ◽  
Robin Fåhraeus
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Mammalian Cells ◽  
Rna World ◽  
Genetic Diseases ◽  
Protein Protein Interactions ◽  
Activation Domain ◽  
Interacting Protein ◽  
Protein Motifs ◽  
Synonymous Mutations

Abstract Structured RNA regulatory motifs exist from the prebiotic stages of the RNA world to the more complex eukaryotic systems. In cases where a functional RNA structure is within the coding sequence a selective pressure drives a parallel co-evolution of the RNA structure and the encoded peptide domain. The p53-MDM2 axis, describing the interactions between the p53 tumor suppressor and the MDM2 E3 ubiquitin ligase, serves as particularly useful model revealing how secondary RNA structures have co-evolved along with corresponding interacting protein motifs, thus having an impact on protein – RNA and protein – protein interactions; and how such structures developed signal-dependent regulation in mammalian systems. The p53(BOX-I) RNA sequence binds the C-terminus of MDM2 and controls p53 synthesis while the encoded peptide domain binds MDM2 and controls p53 degradation. The BOX-I peptide domain is also located within p53 transcription activation domain. The folding of the p53 mRNA structure has evolved from temperature-regulated in pre-vertebrates to an ATM kinase signal-dependent pathway in mammalian cells. The protein – protein interaction evolved in vertebrates and became regulated by the same signaling pathway. At the same time the protein - RNA and protein - protein interactions evolved, the p53 trans-activation domain progressed to become integrated into a range of cellular pathways. We discuss how a single synonymous mutation in the BOX-1, the p53(L22 L), observed in a chronic lymphocyte leukaemia patient, prevents the activation of p53 following DNA damage. The concepts analysed and discussed in this review may serve as a conceptual mechanistic paradigm of the co-evolution and function of molecules having roles in cellular regulation, or the aetiology of genetic diseases and how synonymous mutations can affect the encoded protein.

scholarly journals RNA–protein interactions: disorder, moonlighting and junk contribute to eukaryotic complexity

Open Biology ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 190096 ◽  
Author(s):  
Anna Balcerak ◽  
Alicja Trebinska-Stryjewska ◽  
Ryszard Konopinski ◽  
Maciej Wakula ◽  
Ewa Anna Grzybowska
Keyword(s):  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Binding Motifs ◽  
Coding Regions ◽  
Regulatory Circuits ◽  
Proteins Interactions

RNA–protein interactions are crucial for most biological processes in all organisms. However, it appears that the complexity of RNA-based regulation increases with the complexity of the organism, creating additional regulatory circuits, the scope of which is only now being revealed. It is becoming apparent that previously unappreciated features, such as disordered structural regions in proteins or non-coding regions in DNA leading to higher plasticity and pliability in RNA–protein complexes, are in fact essential for complex, precise and fine-tuned regulation. This review addresses the issue of the role of RNA–protein interactions in generating eukaryotic complexity, focusing on the newly characterized disordered RNA-binding motifs, moonlighting of metabolic enzymes, RNA-binding proteins interactions with different RNA species and their participation in regulatory networks of higher order.

scholarly journals Systematic Identification of Protein Phosphorylation-Mediated Interactions

2020 ◽  
Author(s):  
Brendan M. Floyd ◽  
Kevin Drew ◽  
Edward M. Marcotte
Keyword(s):  
Mass Spectrometry ◽  
Protein Phosphorylation ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Protein ◽  
Mass Spectrometry Data ◽  
Size Exclusion ◽  
Functional Protein

ABSTRACTProtein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

scholarly journals Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

2021 ◽  
Author(s):  
Omer Ziv ◽  
Svetlana Farberov ◽  
Jian You Lau ◽  
Eric A Miska ◽  
Grzegorz Kudla ◽  
...  
Keyword(s):  
Rna Structure ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Structural Domain ◽  
Mrna Targets ◽  
Structural Principles ◽  
Pumilio Proteins

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

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

2019 ◽  
Vol 20 (1) ◽  
pp. 139 ◽  
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.

scholarly journals The Subcellular Distribution of an RNA Quality Control Protein, the Ro Autoantigen, Is Regulated by Noncoding Y RNA Binding

2009 ◽  
Vol 20 (5) ◽  
pp. 1555-1564 ◽  
Author(s):  
Soyeong Sim ◽  
David E. Weinberg ◽  
Gabriele Fuchs ◽  
Keum Choi ◽  
Jina Chung ◽  
...  
Keyword(s):  
Quality Control ◽  
Uv Irradiation ◽  
Subcellular Distribution ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Nuclear Accumulation ◽  
Chimeric Proteins ◽  
Rna Quality Control ◽  
Control Protein

The Ro autoantigen is a ring-shaped RNA-binding protein that binds misfolded RNAs in nuclei and is proposed to function in quality control. In the cytoplasm, Ro binds noncoding RNAs, called Y RNAs, that inhibit access of Ro to other RNAs. Ro also assists survival of mammalian cells and at least one bacterium after UV irradiation. In mammals, Ro undergoes dramatic localization changes after UV irradiation, changing from mostly cytoplasmic to predominantly nuclear. Here, we report that a second role of Y RNAs is to regulate the subcellular distribution of Ro. A mutant Ro protein that does not bind Y RNAs accumulates in nuclei. Ro also localizes to nuclei when Y RNAs are depleted. By assaying chimeric proteins in which portions of mouse Ro were replaced with bacterial Ro sequences, we show that nuclear accumulation of Ro after irradiation requires sequences that overlap the Y RNA binding site. Ro also accumulates in nuclei after oxidative stress, and similar sequences are required. Together, these data reveal that Ro contains a signal for nuclear accumulation that is masked by a bound Y RNA and suggest that Y RNA binding may be modulated during cell stress.

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