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

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22179416 ◽

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.

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Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911892116 ◽

2019 ◽

Vol 116 (42) ◽

pp. 21274-21284 ◽

Cited By ~ 21

Author(s):

Dingquan Huang ◽

Yanbiao Sun ◽

Zhiming Ma ◽

Meiyu Ke ◽

Yong Cui ◽

...

Keyword(s):

Salicylic Acid ◽

Lipid Raft ◽

Plant Pathogens ◽

Regulatory Protein ◽

Cell Communication ◽

Direct Interaction ◽

Membrane Lipid ◽

Dependent Manner ◽

Transmission Electron ◽

Lipid Organization

Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

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Widespread FUS mislocalization is a molecular hallmark of ALS

10.1101/491787 ◽

2019 ◽

Cited By ~ 1

Author(s):

Giulia E. Tyzack ◽

Raphaelle Luisier ◽

Doaa M. Taha ◽

Jacob Neeves ◽

Miha Modic ◽

...

Keyword(s):

Motor Neurons ◽

Nuclear Export ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intron Retention ◽

Direct Interaction ◽

Spinal Cord Tissue ◽

Cytoplasmic Inclusions ◽

Sporadic Als ◽

Fus Protein

AbstractAmyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins (RBPs), which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RBPs mislocalize without necessarily becoming constituents of ALS cytoplasmic inclusions themselves remains unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RBP Fused in Sarcoma (FUS), in an unaggregated state, may occur more widely in ALS that previously recognized. To address this hypothesis, we analysed motor neurons (MNs) from an human ALS induced-pluripotent stem cells (iPSC) model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron-retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

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An interaction network of RNA-binding proteins involved in Drosophila oogenesis

10.1101/2020.01.08.899146 ◽

2020 ◽

Author(s):

Prashali Bansal ◽

Johannes Madlung ◽

Kristina Schaaf ◽

Boris Macek ◽

Fulvia Bono

Keyword(s):

Translational Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Direct Interaction ◽

Interaction Network ◽

Splicing Factors ◽

Mrna Regulation ◽

Interaction Partners ◽

Maternal Transcripts

AbstractDuring Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

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Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

10.1101/2020.11.17.387456 ◽

2020 ◽

Author(s):

Isioma I.I. Enwerem ◽

Nathan D. Elrod ◽

Chung-Te Chang ◽

Ai Lin ◽

Ping Ji ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Molecular Model ◽

Direct Interaction ◽

Vertebrate Development ◽

Regulate Gene Expression ◽

Mrna Decapping ◽

Specific Mrnas ◽

Decapping Enzyme ◽

Pumilio Proteins

AbstractPumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.

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An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra119.001912 ◽

2020 ◽

Vol 19 (9) ◽

pp. 1485-1502

Author(s):

Prashali Bansal ◽

Johannes Madlung ◽

Kristina Schaaf ◽

Boris Macek ◽

Fulvia Bono

Keyword(s):

Translational Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Direct Interaction ◽

Interaction Network ◽

Splicing Factors ◽

Mrna Regulation ◽

Interaction Partners ◽

Maternal Transcripts

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

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Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression

Scientific Reports ◽

10.1038/s41598-021-81429-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Silvia Parisi ◽

Daniela Castaldo ◽

Silvia Piscitelli ◽

Chiara D’Ambrosio ◽

Giuseppina Divisato ◽

...

Keyword(s):

Gene Expression ◽

Dna Methyltransferase ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Binding Protein ◽

Embryonic Stem ◽

Direct Interaction ◽

Mirna Biogenesis ◽

Dna Methyltransferase 3A

AbstractLin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity.

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Musashi1 Contribution to Glioblastoma Development via Regulation of a Network of DNA Replication, Cell Cycle and Division Genes

Cancers ◽

10.3390/cancers13071494 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1494

Author(s):

Mirella Baroni ◽

Caihong Yi ◽

Saket Choudhary ◽

Xiufen Lei ◽

Adam Kosti ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genomic Analysis ◽

Direct Interaction ◽

Combination Strategy ◽

Increased Sensitivity ◽

Gene Expression Alterations ◽

Regulatory Functions

RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1’s core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

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Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus

Journal of Cell Science ◽

10.1242/jcs.108.1.257 ◽

1995 ◽

Vol 108 (1) ◽

pp. 257-263 ◽

Cited By ~ 2

Author(s):

C. Dargemont ◽

M.S. Schmidt-Zachmann ◽

L.C. Kuhn

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Covalent Binding ◽

Mrna Export ◽

Substantial Reduction ◽

Direct Interaction ◽

Transport Pathway ◽

Novel Approach

Primary transcripts in eukaryotic cells undergo several processing steps within the nucleus, and resulting mature RNA molecules are selectively exported to the cytoplasm. Nucleo-cytoplasmic mRNA transport is an active process that likely involves RNA-protein interactions. To identify specific RNA-binding proteins, we designed a novel approach, which allows the analysis of interactions between mRNAs and proteins along the transport pathway. The method consists of inducing in vivo a covalent binding between nuclear proteins and microinjected mRNAs. Using such a procedure, we were able to detect a direct interaction between nucleoporin p62 with mRNA during export. The formation of the mRNA-p62 complex was inhibited by wheat-germ agglutinin, an inhibitor of mRNA export. Antibodies directed against p62 caused a substantial reduction in the rate of mRNA export from the nucleus.

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Cyp33 Binds AU-Rich RNA Motifs via an Extended Interface that Competitively Disrupts the Gene Repressive Cyp33-MLL1 Interaction in vitro

10.1101/2020.08.07.241166 ◽

2020 ◽

Author(s):

Neil R. Lloyd ◽

Deborah S. Wuttke

Keyword(s):

Chromatin Remodeling ◽

Rna Binding ◽

Gene Activation ◽

Direct Interaction ◽

Rna Aptamers ◽

Binding Motif ◽

Rna Recognition Motif ◽

Isomerase Activity ◽

Rrm Domain

AbstractCyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.

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