scholarly journals Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

2015 ◽  
Vol 89 (6) ◽  
pp. 3430-3437 ◽  
Author(s):  
Luana de Borba ◽  
Sergio M. Villordo ◽  
Nestor G. Iglesias ◽  
Claudia V. Filomatori ◽  
Leopoldo G. Gebhard ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Untranslated Region ◽  
Rna Binding ◽  
Rna Folding ◽  
Virus Genome ◽  
Binding Assays ◽  
Functional Studies ◽  
Chemical Probing ◽  
Dumbbell Structure

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified newcis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3′ untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

scholarly journals Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

2005 ◽  
Vol 79 (11) ◽  
pp. 6631-6643 ◽  
Author(s):  
Diego E. Alvarez ◽  
María F. Lodeiro ◽  
Silvio J. Ludueña ◽  
Lía I. Pietrasanta ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Virus Genome ◽  
Viral Rna ◽  
Physical Interaction ◽  
Virus Rna ◽  
Rna Elements ◽  
Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

scholarly journals PERAN GEN STRUKTURAL, GEN NON-STRUKTURAL, DAN UNTRANSLATED REGION DALAM PERAKITAN VIRUS DENGUE

2015 ◽  
Vol 7 (2) ◽  
Author(s):  
Meiranty C. Pangerapan ◽  
Beivy J. Kolondam
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Dengue Fever ◽  
Aedes Albopictus ◽  
Untranslated Region ◽  
Rna Virus ◽  
Virus Genome ◽  
Interferon Response ◽  
Endoplasmic Reticulum Membrane ◽  
Structural Genes

Abstract: Dengue virus is a single-stranded RNA virus that belongs to Flaviviridae family. This virus causes dengue fever which is transmitted by Aedes aegypti dan Aedes albopictus. There are four serotypes of dengue virus; all of them can cause dengue fever. Understanding the genomics of dengue virus is important for research and diagnostics. The genome of dengue virus is 11 kilo-base long. It consists of 5’-untranslated region (5’-UTR), three structural genes (coding capsid protein, pre-membrane/membrane, and envelope), seven non-structural genes (coding NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) and 3’-UTR. Non-structural genes are encoding proteins of viral RNA replication, interferon response, viral assembly and secretion, endoplasmic reticulum membrane invagination induction, immune-mediator induction, and RNA 5’-caping.Keywords: dengue virus, genome, structural genes, non-structural genes, untranslated region.Abstrak: Virus dengue merupakan virus RNA beruntai tunggal yang termasuk dalam famili Flaviviridae. Virus ini adalah penyebab penyakit demam berdarah dengue yang ditransmisikan melalui nyamuk Aedes aegypti dan Aedes albopictus. Ada empat serotipe virus dengue yang telah dikenal secara luas yang ada semuanya dapat menimbulkan penyakit demam berdarah. Pemahaman tentang genomik virus dengue sangat penting untuk pengembangan penelitian dan juga untuk keperluan diagnostik. Genom virus dengue memiliki panjang 11 kilo basa. Genomnya tersusun atas 5’-untranslated region (5’-UTR), tiga gen struktural (mengodekan protein kapsid, premembran/membran dan amplop), tujuh gen non-struktural (mengodekan protein NS1, NS2A, NS2B, NS3, NS4A, NS4B dan NS5) dan 3’-UTR. Gen-gen non-struktural mengodekan protein untuk replikasi RNA virus, respon interferon, perakitan, sekresi partikel virus, menginduksi invaginasi membran retikulum endoplasma, induksi imunomediator dan penambahan tudung pada ujung 5’ RNA.Kata kunci: virus dengue, genom, gen struktural, gen non-struktural, untranslated region

scholarly journals Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marcello Clerici ◽  
Marco Faini ◽  
Ruedi Aebersold ◽  
Martin Jinek
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Molecular Architecture ◽  
Binding Assays ◽  
Critical Determinant ◽  
Recognition Mechanism ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Motif Recognition ◽  
Mrna Polyadenylation

3’ polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

scholarly journals Polyamines Regulate c-Myc Translation through Chk2-dependent HuR Phosphorylation

2009 ◽  
Vol 20 (23) ◽  
pp. 4885-4898 ◽  
Author(s):  
Lan Liu ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Xiao ◽  
Peng-Yuan Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Untranslated Region ◽  
Rna Binding ◽  
Molecular Level ◽  
Intestinal Epithelial Cells ◽  
Rna Binding Protein ◽  
Normal Growth ◽  
Intestinal Epithelial ◽  
The Stability ◽  
Insight Into

All mammalian cells depend on polyamines for normal growth and proliferation, but the exact roles of polyamines at the molecular level remain largely unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we show that in rat intestinal epithelial cells (IECs), polyamines enhanced HuR association with the 3′-untranslated region of the c-Myc mRNA by increasing HuR phosphorylation by Chk2, in turn promoting c-Myc translation. Depletion of cellular polyamines inhibited Chk2 and reduced the affinity of HuR for c-Myc mRNA; these effects were completely reversed by addition of the polyamine putrescine or by Chk2 overexpression. In cells with high content of cellular polyamines, HuR silencing or Chk2 silencing reduced c-Myc translation and c-Myc expression levels. Our findings demonstrate that polyamines regulate c-Myc translation in IECs through HuR phosphorylation by Chk2 and provide new insight into the molecular functions of cellular polyamines.

scholarly journals Dengue virus strain 2 capsid protein switches the annealing pathway and reduces intrinsic dynamics of the conserved 5’ untranslated region

RNA Biology ◽  
2021 ◽  
pp. 1-14
Author(s):  
Xin Ee Yong ◽  
Palur Venkata Raghuvamsi ◽  
Ganesh S. Anand ◽  
Thorsten Wohland ◽  
Kamal K. Sharma
Keyword(s):  
Dengue Virus ◽  
Capsid Protein ◽  
Virus Strain ◽  
Untranslated Region

scholarly journals The RNA Architecture of the SARS-CoV-2 3′-Untranslated Region

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1473
Author(s):  
Junxing Zhao ◽  
Jianming Qiu ◽  
Sadikshya Aryal ◽  
Jennifer L. Hackett ◽  
Jingxin Wang
Keyword(s):  
Untranslated Region ◽  
Dimethyl Sulfate ◽  
Nonstructural Proteins ◽  
Stem Loop ◽  
Cis Acting ◽  
Chemical Probing ◽  
Mutational Profiling ◽  
Genome Transcription ◽  
Polymerase Binding ◽  
Transcription And Replication

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

scholarly journals RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xia Wang ◽  
Jin Wang ◽  
Yu-Man Tsui ◽  
Chaoran Shi ◽  
Ying Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mrna Stability ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Molecular Characteristics ◽  
Specific Gene ◽  
Functional Studies ◽  
Poor Differentiation ◽  
Development In Vitro

AbstractGrowing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

scholarly journals Identification and characterization of a 44 kDa protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization

1996 ◽  
Vol 313 (3) ◽  
pp. 1029-1037 ◽  
Author(s):  
Olivier GENESTE ◽  
Françoise RAFFALLI ◽  
Matti A. LANG
Keyword(s):  
Half Life ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Blocking Agent ◽  
Subcellular Fractionation ◽  
Mrna Stabilization ◽  
Nuclear Extracts

Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041–8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA–protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDA RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.

scholarly journals Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

2005 ◽  
Vol 393 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Catherine Martel ◽  
Paolo Macchi ◽  
Luc Furic ◽  
Michael A. Kiebler ◽  
Luc Desgroseillers
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Nuclear Trafficking ◽  
Binding Domains ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

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