scholarly journals Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

2021 ◽  
Vol 22 (17) ◽  
pp. 9416
Author(s):  
Rafał Mańka ◽  
Pawel Janas ◽  
Karolina Sapoń ◽  
Teresa Janas ◽  
Tadeusz Janas
Keyword(s):  
Lipid Rafts ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Membrane Lipid ◽  
Rna Aptamers ◽  
Target Cells ◽  
Rna Motifs ◽  
Binding Motifs ◽  
Rna Exosome ◽  
Rna Interaction

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.

scholarly journals Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells

2020 ◽  
Vol 21 (22) ◽  
pp. 8503
Author(s):  
Teresa Janas ◽  
Pawel Janas ◽  
Karolina Sapoń ◽  
Tadeusz Janas
Keyword(s):  
Lipid Raft ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Membrane Lipid ◽  
Rna Aptamers ◽  
Region Selection ◽  
Exosomal Mirnas ◽  
Liposomal Membranes ◽  
Rna Complex

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.

scholarly journals Discovery of allele-specific protein-RNA interactions in human transcriptomes

2018 ◽  
Author(s):  
Emad Bahrami-Samani ◽  
Yi Xing
Keyword(s):  
Genetic Variants ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Protein ◽  
Computational Method ◽  
Joint Analysis ◽  
Transcriptional Level ◽  
Rna Seq ◽  
Allele Specific ◽  
Rna Interaction

AbstractGene expression is tightly regulated at the post-transcriptional level through splicing, transport, translation, and decay. RNA-binding proteins (RBPs) play key roles in post-transcriptional gene regulation, and genetic variants that alter RBP-RNA interactions can affect gene products and functions. We developed a computational method ASPRIN (Allele-Specific Protein-RNA Interaction), that uses a joint analysis of CLIP-seq (cross-linking and immunoprecipitation followed by high-throughput sequencing) and RNA-seq data to identify genetic variants that alter RBP-RNA interactions by directly observing the allelic preference of RBP from CLIP-seq experiments as compared to RNA-seq. We used ASPRIN to systematically analyze CLIP-seq and RNA-seq data for 166 RBPs in two ENCODE (Encyclopedia of DNA Elements) cell lines. ASPRIN identified genetic variants that alter RBP-RNA interactions by modifying RBP binding motifs within RNA. Moreover, through an integrative ASPRIN analysis with population-scale RNA-seq data, we showed that ASPRIN can help reveal potential causal variants that affect alternative splicing via allele-specific protein-RNA interactions.

scholarly journals RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants

2021 ◽  
Vol 9 ◽  
Author(s):  
Tao Wang ◽  
Xiaojun Li ◽  
Xiaojing Zhang ◽  
Qing Wang ◽  
Wenqian Liu ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Future Research ◽  
Rna Transport ◽  
Long Distance ◽  
Rna Motifs ◽  
Related Sequence ◽  
Long Distance Transport ◽  
Nucleotide Mutation ◽  
Distance Transport

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.

scholarly journals A deep learning framework to predict binding preference of RNA constituents on protein surface

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jordy Homing Lam ◽  
Yu Li ◽  
Lizhe Zhu ◽  
Ramzan Umarov ◽  
Hanlun Jiang ◽  
...  
Keyword(s):  
Deep Learning ◽  
Protein Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Preference ◽  
Rna Backbone ◽  
Binding Pockets ◽  
Rna Interaction

Abstract Protein-RNA interaction plays important roles in post-transcriptional regulation. However, the task of predicting these interactions given a protein structure is difficult. Here we show that, by leveraging a deep learning model NucleicNet, attributes such as binding preference of RNA backbone constituents and different bases can be predicted from local physicochemical characteristics of protein structure surface. On a diverse set of challenging RNA-binding proteins, including Fem-3-binding-factor 2, Argonaute 2 and Ribonuclease III, NucleicNet can accurately recover interaction modes discovered by structural biology experiments. Furthermore, we show that, without seeing any in vitro or in vivo assay data, NucleicNet can still achieve consistency with experiments, including RNAcompete, Immunoprecipitation Assay, and siRNA Knockdown Benchmark. NucleicNet can thus serve to provide quantitative fitness of RNA sequences for given binding pockets or to predict potential binding pockets and binding RNAs for previously unknown RNA binding proteins.

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 RNA regulons and the RNA-protein interaction network

2012 ◽  
Vol 3 (5) ◽  
pp. 403-414 ◽  
Author(s):  
Jochen Imig ◽  
Alexander Kanitz ◽  
André P. Gerber
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Interaction Network ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Interaction Network ◽  
Coordinated Control ◽  
Non Coding Rnas ◽  
Rna Interaction

AbstractThe development of genome-wide analysis tools has prompted global investigation of the gene expression program, revealing highly coordinated control mechanisms that ensure proper spatiotemporal activity of a cell’s macromolecular components. With respect to the regulation of RNA transcripts, the concept of RNA regulons, which – by analogy with DNA regulons in bacteria – refers to the coordinated control of functionally related RNA molecules, has emerged as a unifying theory that describes the logic of regulatory RNA-protein interactions in eukaryotes. Hundreds of RNA-binding proteins and small non-coding RNAs, such as microRNAs, bind to distinct elements in target RNAs, thereby exerting specific and concerted control over posttranscriptional events. In this review, we discuss recent reports committed to systematically explore the RNA-protein interaction network and outline some of the principles and recurring features of RNA regulons: the coordination of functionally related mRNAs through RNA-binding proteins or non-coding RNAs, the modular structure of its components, and the dynamic rewiring of RNA-protein interactions upon exposure to internal or external stimuli. We also summarize evidence for robust combinatorial control of mRNAs, which could determine the ultimate fate of each mRNA molecule in a cell. Finally, the compilation and integration of global protein-RNA interaction data has yielded first insights into network structures and provided the hypothesis that RNA regulons may, in part, constitute noise ‘buffers’ to handle stochasticity in cellular transcription.

scholarly journals Functionally related transcripts have common RNA motifs for specific RNA-binding proteins in trypanosomes

2008 ◽  
Vol 9 (1) ◽  
pp. 107 ◽  
Author(s):  
Griselda Noé ◽  
Javier G De Gaudenzi ◽  
Alberto C Frasch
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Motifs

scholarly journals Cholesterol Dependence of Varicella-Zoster Virion Entry into Target Cells

2007 ◽  
Vol 81 (14) ◽  
pp. 7548-7558 ◽  
Author(s):  
S. Hambleton ◽  
S. P. Steinberg ◽  
M. D. Gershon ◽  
A. A. Gershon
Keyword(s):  
Lipid Rafts ◽  
Viral Entry ◽  
Membrane Lipid ◽  
Viral Envelope ◽  
Target Cells ◽  
Cholesterol Depletion ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Focus Formation ◽  
Varicella Zoster

ABSTRACT The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-β-cyclodextrin (MβCD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by MβCD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.

scholarly journals CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion

2021 ◽  
Author(s):  
Alexander Krohannon ◽  
Mansi Srivastava ◽  
Simone Rauch ◽  
Rajneesh Srivastava ◽  
Bryan Dickinson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Training Data ◽  
Therapeutic Interventions ◽  
Hek293 Cells ◽  
Data Set ◽  
Guide Rna ◽  
Target Transcript ◽  
Protein Occupancy ◽  
Rna Interaction

Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which further limit the scope of effective target sequences. As a result, there currently exists no method to predict whether a specific sgRNA will effectively knockdown a transcript. Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information on RNA. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in Hela cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.

scholarly journals Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

2020 ◽  
Author(s):  
Clémentine Delan-Forino ◽  
Christos Spanos ◽  
Juri Rappsilber ◽  
David Tollervey
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Rna Helicase ◽  
Nuclear Rna ◽  
Rna Exosome

ABSTRACTDuring nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

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