scholarly journals The Structure of Human Parechovirus 1 Reveals an Association of the RNA Genome with the Capsid

2015 ◽  
Vol 90 (3) ◽  
pp. 1377-1386 ◽  
Author(s):  
Sergei Kalynych ◽  
Lenka Pálková ◽  
Pavel Plevka
Keyword(s):  
Small Molecules ◽  
Capsid Protein ◽  
Protein Interactions ◽  
Rna Binding ◽  
Particle Surface ◽  
Gastrointestinal Disorders ◽  
Human Parechovirus ◽  
Antiviral Compounds ◽  
Rna Genome

ABSTRACTParechoviruses are human pathogens that cause diseases ranging from gastrointestinal disorders to encephalitis. Unlike those of most picornaviruses, parechovirus capsids are composed of only three subunits: VP0, VP1, and VP3. Here, we present the structure of a human parechovirus 1 (HPeV-1) virion determined to a resolution of 3.1 Å. We found that interactions among pentamers in the HPeV-1 capsid are mediated by the N termini of VP0s, which correspond to the capsid protein VP4 and the N-terminal part of the capsid protein VP2 of other picornaviruses. In order to facilitate delivery of the virus genome into the cytoplasm, the N termini of VP0s have to be released from contacts between pentamers and exposed at the particle surface, resulting in capsid disruption. A hydrophobic pocket, which can be targeted by capsid-binding antiviral compounds in many other picornaviruses, is not present in HPeV-1. However, we found that interactions between the HPeV-1 single-stranded RNA genome and subunits VP1 and VP3 in the virion impose a partial icosahedral ordering on the genome. The residues involved in RNA binding are conserved among all parechoviruses, suggesting a putative role of the genome in virion stability or assembly. Therefore, putative small molecules that could disrupt HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors.IMPORTANCEHuman parechoviruses (HPeVs) are pathogens that cause diseases ranging from respiratory and gastrointestinal disorders to encephalitis. Recently, there have been outbreaks of HPeV infections in Western Europe and North America. We present the first atomic structure of parechovirus HPeV-1 determined by X-ray crystallography. The structure explains why HPeVs cannot be targeted by antiviral compounds that are effective against other picornaviruses. Furthermore, we found that the interactions of the HPeV-1 genome with the capsid resulted in a partial icosahedral ordering of the genome. The residues involved in RNA binding are conserved among all parechoviruses, suggesting an evolutionarily fixed role of the genome in virion assembly. Therefore, putative small molecules disrupting HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors.

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 Dissecting the Role of Circular RNAs in Sarcomas with Emphasis on Osteosarcomas

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1642
Author(s):  
Eleftheria Lakiotaki ◽  
Dimitrios S. Kanakoglou ◽  
Andromachi Pampalou ◽  
Eleni A. Karatrasoglou ◽  
Christina Piperi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Malignant Tumors ◽  
Kaposi Sarcoma ◽  
Metastatic Potential ◽  
Circular Rnas ◽  
Mesenchymal Tumors ◽  
Protein Coding ◽  
Ongoing Research

Circular RNAs (circRNAs) are single-stranded RNAs generated from exons back-splicing from a single pre-mRNA, forming covalently closed loop structures which lack 5′-3′-polarity or polyadenylated tail. Ongoing research depicts that circRNAs play a pivotal role in tumorigenesis, tumor progression, metastatic potential and chemoresistance by regulating transcription, microRNA (miRNA) sponging, RNA-binding protein interactions, alternative splicing and to a lesser degree, protein coding. Sarcomas are rare malignant tumors stemming from mesenchymal cells. Due to their clinically insidious onset, they often present at advanced stage and their treatment may require aggressive chemotherapeutic or surgical options. This review is mainly focused on the regulatory functions of circRNAs on osteosarcoma progression and their potential role as biomarkers, an area which has prompted lately extensive research. The attributed oncogenic role of circRNAs on other mesenchymal tumors such as Kaposi Sarcoma (KS), Rhabdomyosarcoma (RMS) or Gastrointestinal Stromal Tumors (GISTs) is also described. The involvement of circRNAs on sarcoma oncogenesis and relevant emerging diagnostic, prognostic and therapeutic applications are expected to gain more research interest in the future.

scholarly journals Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

1998 ◽  
Vol 18 (2) ◽  
pp. 676-684 ◽  
Author(s):  
Wan-Jiang Zhang ◽  
Jane Y. Wu
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Interacting Protein

ABSTRACT Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.

scholarly journals Identification of protein-protein and ribonucleoprotein complexes containing Hfq

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joël Caillet ◽  
Bruno Baron ◽  
Irina V. Boni ◽  
Célia Caillet-Saguy ◽  
Eliane Hajnsdorf
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Direct Interaction ◽  
Rnase A ◽  
Rnase E ◽  
Protein Protein Interactions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes

Abstract Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

scholarly journals The role of disorder in RNA binding affinity and specificity

Open Biology ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 200328
Author(s):  
Diana S. M. Ottoz ◽  
Luke E. Berchowitz
Keyword(s):  
Protein Interactions ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Multiple Partners ◽  
Cellular Environment ◽  
Rna Targets

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.

Understanding the Role of Flavonoid Based Small Molecules in Modulating the Oncogenic Protein-Protein Interactions: A Quest for Therapeutic Arsenal

2022 ◽  
Vol 1248 ◽  
pp. 131511
Author(s):  
Abhijit Karmakar ◽  
Tamanna Mallick ◽  
Chandrani Fouzder ◽  
Alpana Mukhuty ◽  
Samiran Mondal ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Oncogenic Protein

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.

scholarly journals Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

2014 ◽  
Vol 19 (8) ◽  
pp. 1147-1153 ◽  
Author(s):  
Shaun P. Falk ◽  
Bernard Weisblum
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Drug Target ◽  
Rna Binding ◽  
Yellow Fever Virus ◽  
Mrna Transcript ◽  
Mobility Shift ◽  
Adenosyl Methionine ◽  
Mobility Shift Assay

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5′ end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a Kd = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50 for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5′-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z′ of 0.83 and was successfully used to screen a library of known bioactives.

scholarly journals Small Molecule Modulation of MEMO1 Protein-Protein Interactions

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1031-A1031
Author(s):  
Julie A Pollock ◽  
Courtney L Labrecque ◽  
Cassidy N Hilton ◽  
Justin Airas ◽  
Alexis Blake ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Small Molecules ◽  
Protein Interactions ◽  
Scaffolding Protein ◽  
Breast Cancer Cell Lines ◽  
Protein Protein Interactions ◽  
Tyrosine Residues ◽  
Fluorescence Polarization Assay ◽  
Computational Analyses

Abstract MEMO1 (mediator of ErbB2-driven cell motility) is upregulated in breast tumors and has been correlated with poor prognosis in patients. As a scaffolding protein that binds to phosphorylated-tyrosine residues on receptors such as estrogen receptor and ErbB2, MEMO1 levels can influence phosphorylation cascades. Using our previously developed fluorescence polarization assay, we have identified small molecules with the ability to disrupt the interactions of MEMO1. We have performed limited structure-activity-relationship studies and computational analyses to investigate the molecular requirements for MEMO1 inhibition. The most promising compounds exhibit slowed migration of breast cancer cell lines (T47D and SKBR3) in a wound-healing assay emulating results obtained from the knockdown of MEMO1 protein. To our knowledge, these are the first small molecules targeting the MEMO1 protein-protein interface and therefore, will be invaluable tools for the investigation of the role of the MEMO1 in breast cancer and other biological contexts.

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