Molecular Mechanism of SARS-CoVs Orf6 Targeting the Rae1–Nup98 Complex to Compete With mRNA Nuclear Export

The accessory protein Orf6 is uniquely expressed in sarbecoviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is an ongoing pandemic. SARS-CoV-2 Orf6 antagonizes host interferon signaling by inhibition of mRNA nuclear export through its interactions with the ribonucleic acid export 1 (Rae1)–nucleoporin 98 (Nup98) complex. Here, we confirmed the direct tight binding of Orf6 to the Rae1-Nup98 complex, which competitively inhibits RNA binding. We determined the crystal structures of both SARS-CoV-2 and SARS-CoV-1 Orf6 C-termini in complex with the Rae1–Nup98 heterodimer. In each structure, SARS-CoV Orf6 occupies the same potential mRNA-binding groove of the Rae1–Nup98 complex, comparable to the previously reported structures of other viral proteins complexed with Rae1-Nup98, indicating that the Rae1–Nup98 complex is a common target for different viruses to impair the nuclear export pathway. Structural analysis and biochemical studies highlight the critical role of the highly conserved methionine (M58) of SARS-CoVs Orf6. Altogether our data unravel a mechanistic understanding of SARS-CoVs Orf6 targeting the mRNA-binding site of the Rae1–Nup98 complex to compete with the nuclear export of host mRNA, which further emphasizes that Orf6 is a critical virulence factor of SARS-CoVs.

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Critical role of tristetraprolin and AU‐rich element RNA‐binding protein 1 in the suppression of cancer cell growth by globular adiponectin

FEBS Open Bio ◽

10.1002/2211-5463.12541 ◽

2018 ◽

Vol 8 (12) ◽

pp. 1964-1976 ◽

Cited By ~ 1

Author(s):

Nirmala Tilija Pun ◽

Amrita Khakurel ◽

Aastha Shrestha ◽

Sang‐Hyun Kim ◽

Pil‐Hoon Park

Keyword(s):

Cell Growth ◽

Cancer Cell ◽

Rna Binding ◽

Binding Protein ◽

Critical Role ◽

Rna Binding Protein ◽

Cancer Cell Growth ◽

Globular Adiponectin

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Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007774117 ◽

2020 ◽

Vol 117 (43) ◽

pp. 26719-26727

Author(s):

Han Feng ◽

Huabin Tian ◽

Yong Wang ◽

Qixiang Zhang ◽

Ni Lin ◽

...

Keyword(s):

Molecular Mechanism ◽

Nuclear Export ◽

Rna Binding ◽

Mrna Export ◽

Direct Interaction ◽

Selective Inhibition ◽

Murine Gammaherpesvirus ◽

Binding Groove ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1–Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1–Nup98, the ORF10–Rae1–Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10–RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1–Nup98 in regulating host mRNA export.

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Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains

Biological Chemistry ◽

10.1515/hsz-2013-0111 ◽

2013 ◽

Vol 394 (8) ◽

pp. 1077-1090 ◽

Cited By ~ 45

Author(s):

Kristin Wächter ◽

Marcel Köhn ◽

Nadine Stöhr ◽

Stefan Hüttelmaier

Keyword(s):

Subcellular Localization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Structural Constraints ◽

Cellular Functions ◽

Dependent Protein ◽

Mrna Binding

Abstract The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

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Abstract P-22: Enhanced Crosslinking and Immunoprecipitation (Eclip) Data Reveal Interactions of RNA Binding Proteins with the Human Ribosome

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p22 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S21-S21

Author(s):

Andrey Buyan ◽

Ivan Kulakovskiy ◽

Sergey Dmitriev

Keyword(s):

Translational Control ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Repetitive Sequences ◽

Structural Data

Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.

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UHRF1 Suppresses HIV-1 Transcription and Promotes HIV-1 Latency by Competing with p-TEFb for Ubiquitination-Proteasomal Degradation of Tat

mBio ◽

10.1128/mbio.01625-21 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Taizhen Liang ◽

Qiao Zhang ◽

Ziyao Wu ◽

Pei Chen ◽

Yifan Huang ◽

...

Keyword(s):

Viral Protein ◽

Critical Role ◽

Ring Finger ◽

Proteasomal Degradation ◽

Viral Proteins ◽

Host Factor ◽

Host Factors ◽

Ring Finger Domain ◽

Hiv 1

HIV-1 latency is systematically modulated by host factors and viral proteins. In our work, we identified a critical role of host factor ubiquitin-like with PHD and RING finger domain 1 (UHRF1) in HIV-1 latency via the modulation of the viral protein Tat stability.

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Emerging Role of Circular RNA–Protein Interactions

Non-Coding RNA ◽

10.3390/ncrna7030048 ◽

2021 ◽

Vol 7 (3) ◽

pp. 48

Author(s):

Arundhati Das ◽

Tanvi Sinha ◽

Sharmishtha Shyamal ◽

Amaresh Chandra Panda

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Nuclear Export ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Large Family ◽

Circular Rnas ◽

Protein Interaction Data ◽

Cell Physiology

Circular RNAs (circRNAs) are emerging as novel regulators of gene expression in various biological processes. CircRNAs regulate gene expression by interacting with cellular regulators such as microRNAs and RNA binding proteins (RBPs) to regulate downstream gene expression. The accumulation of high-throughput RNA–protein interaction data revealed the interaction of RBPs with the coding and noncoding RNAs, including recently discovered circRNAs. RBPs are a large family of proteins known to play a critical role in gene expression by modulating RNA splicing, nuclear export, mRNA stability, localization, and translation. However, the interaction of RBPs with circRNAs and their implications on circRNA biogenesis and function has been emerging in the last few years. Recent studies suggest that circRNA interaction with target proteins modulates the interaction of the protein with downstream target mRNAs or proteins. This review outlines the emerging mechanisms of circRNA–protein interactions and their functional role in cell physiology.

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N6-Methyladenosine Modification of PTTG3P Contributes to Colorectal Cancer Proliferation via YAP1

Frontiers in Oncology ◽

10.3389/fonc.2021.669731 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yang Zheng ◽

Yue Wang ◽

Yiyang Liu ◽

Longfei Xie ◽

Jinnian Ge ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Progression ◽

Expression Profiles ◽

Critical Role ◽

Methylation Status ◽

Tumor Model ◽

Loss Of Function ◽

Cancer Proliferation ◽

Mrna Binding

BackgroundLong noncoding RNAs (lncRNAs) have emerged to have irreplaceable roles in the epigenetic regulation of cancer progression, but their biological functions in colorectal cancer (CRC) remain unclear.MethodsLncRNA expression profiles in CRC tissue and their normal counterpart were explored. Through gain and loss of function approaches, the role of lncRNA PTTG3P was validated in relevant CRC cells and subcutaneous tumor model. The correlations of PTTG3P expression with clinical outcomes were assessed.ResultsPTTG3P was upregulated in CRC tissues and was closely correlated with unsatisfactory prognosis. PTTG3P facilitated glycolysis and proliferation, and the transcriptional regulator YAP1 was necessary for PTTG3P-induced proliferation. Mechanistically, the N6-methyladenosine (m6A) subunit METTL3 increased PTTG3P expression by influencing its stability, while insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) could identify PTTG3P m6A methylation status and bind to it. IGF2BP2 knockdown partly recovered PTTG3P expression induced by METTL3, indicating that METTL3-regulated PTTG3P expression depended on the presence of IGF2BP2. Finally, rescue assays validated the critical role of the METTL3/PTTG3P/YAP1 axis on CRC proliferation.ConclusionsPTTG3P is an independent prognostic biomarker for CRC. The METTL3/PTTG3P/YAP1 axis promotes the progression of CRC and is a promising treatment target.

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Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

10.1101/2019.12.18.881649 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer Elliott ◽

Jenna E. Eschbach ◽

Pratibha C. Koneru ◽

Wen Li ◽

Maritza Puray Chavez ◽

...

Keyword(s):

Rna Binding ◽

Critical Role ◽

Underlying Mechanism ◽

Class Ii ◽

Viral Rna ◽

Target Cells ◽

Ribonucleoprotein Complexes ◽

Virion Morphogenesis ◽

Hiv 1

ABSTRACTA large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

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The structure and evolution of eukaryotic chaperonin-containing TCP-1 and its mechanism that folds actin into a protein spring

Biochemical Journal ◽

10.1042/bcj20170378 ◽

2018 ◽

Vol 475 (19) ◽

pp. 3009-3034 ◽

Cited By ~ 13

Author(s):

Keith Robert Willison

Keyword(s):

Atp Hydrolysis ◽

Critical Role ◽

Tight Binding ◽

Size Control ◽

Folding Energy ◽

Control Networks ◽

Double Ring ◽

Sequential Binding ◽

Initial Binding

Actin is folded to its native state in eukaryotic cytosol by the sequential allosteric mechanism of the chaperonin-containing TCP-1 (CCT). The CCT machine is a double-ring ATPase built from eight related subunits, CCT1–CCT8. Non-native actin interacts with specific subunits and is annealed slowly through sequential binding and hydrolysis of ATP around and across the ring system. CCT releases a folded but soft ATP-G-actin monomer which is trapped 80 kJ/mol uphill on the folding energy surface by its ATP-Mg2+/Ca2+ clasp. The energy landscape can be re-explored in the actin filament, F-actin, because ATP hydrolysis produces dehydrated and more compact ADP-actin monomers which, upon application of force and strain, are opened and closed like the elements of a spring. Actin-based myosin motor systems underpin a multitude of force generation processes in cells and muscles. We propose that the water surface of F-actin acts as a low-binding energy, directional waveguide which is recognized specifically by the myosin lever-arm domain before the system engages to form the tight-binding actomyosin complex. Such a water-mediated recognition process between actin and myosin would enable symmetry breaking through fast, low energy initial binding events. The origin of chaperonins and the subsequent emergence of the CCT–actin system in LECA (last eukaryotic common ancestor) point to the critical role of CCT in facilitating phagocytosis during early eukaryotic evolution and the transition from the bacterial world. The coupling of CCT-folding fluxes to the cell cycle, cell size control networks and cancer are discussed together with directions for further research.

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Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin

Biochemical Journal ◽

10.1042/bj3450621 ◽

2000 ◽

Vol 345 (3) ◽

pp. 621-625 ◽

Cited By ~ 5

Author(s):

Martina ANDBERG ◽

Anders WETTERHOLM ◽

Juan F. MEDINA ◽

Jesper Z. HAEGGSTRÖM

Keyword(s):

Epoxide Hydrolase ◽

Critical Role ◽

Tight Binding ◽

Leukotriene B4 ◽

Catalytic Residue ◽

Ic50 Values ◽

Leukotriene A4 Hydrolase ◽

Leukotriene A4 ◽

Structural Determinant

Leukotriene A4 hydrolase is a bifunctional Zn2+-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B4. From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC50 values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A4 hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.

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