scholarly journals Insights into the structure-driven protein interactivity of RNA molecules

2018 ◽  
Author(s):  
Natalia Sanchez de Groot ◽  
Alexandros Armaos ◽  
Ricardo Graña Montes ◽  
Marion Alriquet ◽  
Giulia Calloni ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
High Throughput Sequencing ◽  
Interaction Network ◽  
Recent Experimental Data ◽  
Rna Molecules ◽  
Rna Granules ◽  
Complex Interdependence

ABSTRACTThe 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 that govern interactions in protein-RNA networks are poorly known at present. Here we used the most recent experimental data to investigate the relationship between RNA structure and protein interactions. Our results show that, independently of the particular technique, the amount of structure in RNA molecules correlates with the capacity of binding to proteins in vitro and in vivo. To validate this observation, we generated an in vitro network that mimics the composition of phase-separated RNA granules. We observed that RNA, when structured, competes with protein binding and can rearrange the interaction network. The simplicity of the principle bears great potential to boost the understanding and modelling of cellular processes involving RNA-protein interactions.

scholarly journals A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2020 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B. Fessler ◽  
Craig A. Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

ABSTRACTDue to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art SHAPE reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

scholarly journals CROSSalive: a web server for predicting the in vivo structure of RNA molecules

2019 ◽  
Author(s):  
Riccardo Delli Ponti ◽  
Alexandros Armaos ◽  
Andrea Vandelli ◽  
Gian Gaetano Tartaglia
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Cross Validation ◽  
Supplementary Information ◽  
Supplementary Data ◽  
High Confidence ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Motivation RNA structure is difficult to predict in vivo due to interactions with enzymes and other molecules. Here we introduce CROSSalive, an algorithm to predict the single- and double-stranded regions of RNAs in vivo using predictions of protein interactions. Results Trained on icSHAPE data in presence (m6a+) and absence of N6 methyladenosine modification (m6a-), CROSSalive achieves cross-validation accuracies between 0.70 and 0.88 in identifying high-confidence single- and double-stranded regions. The algorithm was applied to the long non-coding RNA Xist (17 900 nt, not present in the training) and shows an Area under the ROC curve of 0.83 in predicting structured regions. Availability and implementation CROSSalive webserver is freely accessible at http://service.tartaglialab.com/new_submission/crossalive Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2021 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B Fessler ◽  
Craig A Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

Abstract Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

scholarly journals RNA modifications detection by comparative Nanopore direct RNA sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrien Leger ◽  
Paulo P. Amaral ◽  
Luca Pandolfini ◽  
Charlotte Capitanchik ◽  
Federica Capraro ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput Sequencing ◽  
Control Sample ◽  
Analytical Framework ◽  
Rna Modifications ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Experimental Approaches

AbstractRNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

scholarly journals RNA structure through multidimensional chemical mapping

2016 ◽  
Author(s):  
Siqi Tian ◽  
Rhiju Das
Keyword(s):  
Structure Determination ◽  
Rna Structure ◽  
De Novo ◽  
Compensatory Mutation ◽  
Chemical Mapping ◽  
Rna Molecules ◽  
Viral Genomes ◽  
Rna Interaction

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed .OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to two hundred nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches, that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-crosslink-map expansion to overcome this and other current limitations to resolving the in vivo "RNA structurome".

scholarly journals Homeostatic Plasticity Requires Remodeling of the Homer-Shank Interactome

2020 ◽  
Author(s):  
Whitney E. Heavner ◽  
Haley Speed ◽  
Jonathan D. Lautz ◽  
Edward P. Gniffke ◽  
Karen B. Immendorf ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cortical Neurons ◽  
Barrel Cortex ◽  
Protein Complexes ◽  
Sensory Deprivation ◽  
Interaction Network ◽  
Autism Spectrum ◽  
Homeostatic Plasticity

AbstractNeurons maintain constant levels of excitability using homeostatic scaling, which adjusts relative synaptic strength in response to large changes in overall activity. It is still unknown how homeostatic scaling affects network-level protein interactions in the synapse despite extensive reporting of individual scaling-associated transcriptomic and proteomic changes. Here, we assessed a glutamatergic synapse protein interaction network (PIN) composed of 380 binary interactions among 21 protein members to identify protein complexes altered by synaptic scaling in vitro and in vivo. In cultured cortical neurons, we observed widespread bidirectional PIN alterations during up- and downscaling that reflected rapid glutamate receptor shuttling via synaptic scaffold remodeling. Sensory deprivation of the barrel cortex caused a PIN response that reflected changes in mGluR tone and NMDAR-dependent metaplasticity, consistent with emerging models of homeostatic plasticity in the barrel cortex that restore excitatory/inhibitory balance. Mice lacking Homer1 or Shank3B did not undergo normal PIN rearrangements, suggesting that these Autism Spectrum Disorder (ASD)-linked proteins serve as structural hubs for synaptic homeostasis. Our approach demonstrates how changes in the protein content of synapses during homeostatic plasticity translate into functional PIN alterations that mediate changes in neuron excitability.

scholarly journals CROSSalive: a web server for predicting the in vivo structure of RNA molecules

2019 ◽  
Author(s):  
Riccardo Delli Ponti ◽  
Alexandros Armaos ◽  
Gian Gaetano Tartaglia
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Sequence Length ◽  
Test Set ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Map Data ◽  
Long Non Coding Rna

ABSTRACTHere we introduce CROSSalive, an algorithm to predict the RNA secondary structure profile (double and single stranded regions) in vivo and without sequence length limitations. Using predictions of protein interactions CROSSalive predicts the effect of N6 adenosine methylation (m6a) on RNA structure. Trained on icSHAPE data in presence (m6a+) and absence (m6a-) of methylation CROSSalive achieves an accuracy of 0.88 on the test set. The algorithm was also applied to the murine long non-coding RNA Xist (17900 nt, not present in the training) and shows a Pearson’s correlation of 0.45 with SHAPE-map data. CROSSalive webserver is freely accessible at the following page: http://service.tartaglialab.com/new_submission/crossalive

scholarly journals RNA structure through multidimensional chemical mapping

2016 ◽  
Vol 49 ◽  
Author(s):  
Siqi Tian ◽  
Rhiju Das
Keyword(s):  
Structure Determination ◽  
Rna Structure ◽  
De Novo ◽  
Compensatory Mutation ◽  
Chemical Mapping ◽  
Rna Molecules ◽  
Viral Genomes ◽  
Rna Interaction

AbstractThe discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomesin vivo, but the resulting one-dimensional data generally remain too information-poor to allow accuratede novostructure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body ofin vitroblind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving thein vivo ‘RNA structurome’.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

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