scholarly journals Application of the 3'-Untranslated Region of Messenger RNA from Measles Virus Matrix Protein as an RNA Stabilizer: Implications in Pharmaceutical Biotechnology

2019 ◽  
Vol 9 (1) ◽  
pp. 53
Author(s):  
Marzieh Marzbany ◽  
Fatemeh Ghassemi ◽  
Mahsa Rasekhian
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Measles Virus ◽  
Matrix Protein ◽  
Rna Binding ◽  
Rna Stability ◽  
Minimum Free Energy ◽  
Expression Vectors ◽  
Dependent Manner

BACKGROUND: The market for the use of recombinant proteins for medical applications has been increasing in recent years. In many cases including fast production of significant amounts of protein for research purposes, transient transfection is the method of choice. In this regard expression vectors are one of the decisive factors in the cost-effectiveness of the production process. The genetic elements found in the 3’untranslated region (UTR) of mRNA expressed by such vectors, play an essential role in determining its stability and thus in the efficiency of the process. METHODS: In this study, the 3'UTR of matrix protein from the Measles Virus (MV) was used to construct a reporter plasmid containing Enhanced Green Fleurocent Protein (EGFP). The reporter construct was transfected into three cell lines. The effect of 3'UTR on mRNA stability was evaluated by real-time PCR. Secondary structure of the mrna was predicted based on minimum free energy. 3'UTR was analyzed in silico for the presence of binding motifs for trans-acting elements with known effects on RNA stability. RESULTS: Addition of 3’UTR of MV matrix protein sequence to the 3’ end of the mRNA, increased the EGFP- mRNA stability in time and cell-dependent manner. Analysis for the presence of known cis-acting motifs in 3’UTR indicated the presence of two PABPC1 binding sites, an RNA-binding protein, known for its stability and translation enhancing effects. CONCLUSION: Our results verified the potential of the 3'UTR region of matrix protein mRNA for improvement of transient recombinant protein production and vector design for mammalian cell hosts.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2111-2120 ◽  
Author(s):  
Maria F. Czyzyk-Krzeska ◽  
Amy C. Bendixen
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mobility Shift ◽  
Protein Factor ◽  
Ribonucleoprotein Complexes ◽  
Sds Page

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3′ untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as CP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

scholarly journals RNA-Binding Protein IGF2BP1 Enhances mRNA Stability and Translation Efficiency of INHBA to Promote the Invasion and Migration of Esophageal Squamous Cancer Cells

2021 ◽  
Author(s):  
Juan-Juan Wang ◽  
Ding-Xiong Chen ◽  
Yu Zhang ◽  
Xin Xu ◽  
Yan Cai ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Rna Binding ◽  
Binding Protein ◽  
Mrna Level ◽  
Rna Stability ◽  
Translation Efficiency ◽  
Invasion And Migration ◽  
Squamous Cancer ◽  
And Migration

Abstract BackgroundMetastasis are mainly responsible for the death of patients with advanced esophageal squamous cell carcinoma (ESCC). At present, there is no targeted drug for the treatment of ESCC in clinic practice. The present study aims to investigate the roles and implication of IGF2BP1 overexpression in ESCC.MethodsIGF2BP1 protein expression was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA were analyzed with TCGA datasets and by RNA in situ hybridization (RISH). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the targets and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA-pulldown, immunofluorescence (IF), gene specific m6A PCR and RNA stability assay were used to uncover the molecular mechanism of IGF2BP1 dysregulation. The methylation level of IGF2BP1 promoter region was detected by MSP-PCR. BTYNB, a small molecular inhibitor which could block the binding of IGF2BP1 to c-Myc mRNA, was evaluated for the inhibition effect on the malignant phenotypes of ESCC cells.ResultsIGF2BP1 overexpression was detected in ESCC tissues and associated with depth of tumor invasion. Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis. Importantly, INHBA was identified as a direct target of IGF2BP1 in ESCC cells, which had a role in promoting the malignant phenotypes. TCGA data and RISH analyses showed that the mRNA level of INHBA was upregaluted in ESCC tissues as well. Mechanistically, IGF2BP1 bound and stabilized INHBA mRNA and then enhanced its translation, leading to an activation of Smad2/3 signaling. Ras GTPase-activating protein-binding protein 1 (G3BP1) was recruited by IGF2BP1 to participate in activating the signaling process, which was inhibited by the IGF2BP1 inhibitor BTYNB. Of note, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation.ConclusionsIGF2BP1 overexpression promotes the invasion and migration of ESCC cells by up-regulating TGF-β-Smad2/3 pathway through enhancing INHBA mRNA stability and translation, providing a potential therapeutic target for ESCC treatment.

scholarly journals Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

2021 ◽  
Author(s):  
Sarah E. Hickson ◽  
Eden Brekke ◽  
Johannes Schwerk ◽  
Indraneel Saluhke ◽  
Shivam Zaver ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Structural Features ◽  
Differential Sensitivity ◽  
Restriction Factor ◽  
Dependent Manner ◽  
Tetratricopeptide Repeats ◽  
Rna Interaction

ABSTRACTAlphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5’UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3’ untranslated region (3’UTR) of the virus. We investigated the potential role of variability in the 3’UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3’UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3’UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3’UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3’UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

scholarly journals c-Myc-activated USP2-AS1 suppresses senescence and promotes tumor progression via stabilization of E2F1 mRNA

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Bingyan Li ◽  
Guang Zhang ◽  
Zhongyu Wang ◽  
Yang Yang ◽  
Chenfeng Wang ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Rna Binding ◽  
Noncoding Rnas ◽  
Negative Regulator ◽  
Cancer Initiation ◽  
Important Player ◽  
Oncogenic Function ◽  
Transcriptional Target

AbstractThe c-Myc oncoprotein plays a prominent role in cancer initiation, progression, and maintenance. Long noncoding RNAs (lncRNAs) are recently emerging as critical regulators of the c-Myc signaling pathway. Here, we report the lncRNA USP2-AS1 as a direct transcriptional target of c-Myc. Functionally, USP2-AS1 inhibits cellular senescence and acts as an oncogenic molecule by inducing E2F1 expression. Mechanistically, USP2-AS1 associates with the RNA-binding protein G3BP1 and facilitates the interaction of G3BP1 to E2F1 3′-untranslated region, thereby leading to the stabilization of E2F1 messenger RNA. Furthermore, USP2-AS1 is shown as a mediator of the oncogenic function of c-Myc via the regulation of E2F1. Together, these findings suggest that USP2-AS1 is a negative regulator of cellular senescence and also implicates USP2-AS1 as an important player in mediating c-Myc function.

METTL3 Promotes Oral Squamous Cell Carcinoma Progression by Enhancing SLC7A11 mRNA Stability via m6A-Mediated IGF2BP2 Binding

2021 ◽  
Author(s):  
Le Xu ◽  
Qingxiang Li ◽  
Yifei Wang ◽  
Lin Wang ◽  
Yuxing Guo ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Mrna Stability ◽  
Messenger Rna ◽  
Specific Treatment ◽  
Dependent Manner ◽  
Rna Seq

Abstract Background: As the key enzyme of the N6-methyladenosine (m6A) in eukaryotic messenger RNA, METTL3 plays important roles in tumor progression, but the exact mechanism by which METTL3 controls oral squamous cell carcinoma (OSCC) progression remains unclear. Methods: METTL3 expression in OSCC samples was analyzed by qPCR and immunohistochemistry. The effects of METTL3 suppression on OSCC cell lines were measured by CCK-8, Ki-67 flow cytometry analysis, invasion transwell and wound healing assays. MeRIP-seq and RNA-seq analysis were performed to explore target gene of METTL3. RIP-qPCR and RNA stability assays were performed to explore the mechanism by which METTL3 regulated the target genes. Triptolide was used to evaluate its specific treatment effects on METTL3 in OSCC cells. BALB/c nude mice were used to establish orthotopic and subcutaneous xenograft models to verify the in vitro results.Results: METTL3 was upregulated in OSCC tissues than adjacent normal tissues, and its expression was associated with T stage, lymphatic metastasis and prognosis. In vitro and in vivo studies suggested that METTL3 suppression impaired cell proliferation, invasion, and migration. MeRIP-seq and RNA-seq analysis identified that SLC7A11 mRNA was the m6A target of METTL3, which was verified by meRIP-qPCR, qPCR and western blot. METTL3 depletion decreased the stability of SLC7A11 mRNA, and IGF2BP2 was involved in this process. Moreover, METTL3 knockdown attenuated the binding between SLC7A11 mRNA and IGF2BP2, finally leading to accelerate SLC7A11 mRNA degradation. Triptolide inhibited METTL3 and SLC7A11 expression in a dose-dependent manner, thus suppressing malignancy of OSCC cells. Conclusions: METTL3 enhances the mRNA stability of SLC7A11 via m6A-mediated binding of IGF2BP2, which thus promotes OSCC progression, and triptolide inhibits OSCC by suppressing METTL3-SLC7A11 axis.

scholarly journals miR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels

2010 ◽  
Vol 30 (17) ◽  
pp. 4197-4210 ◽  
Author(s):  
Kotb Abdelmohsen ◽  
Emmette R. Hutchison ◽  
Eun Kyung Lee ◽  
Yuki Kuwano ◽  
Mihee M. Kim ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Neuronal Development ◽  
Rna Binding ◽  
Developmentally Regulated ◽  
Cytoskeleton Organization ◽  
Evolutionarily Conserved ◽  
Mouse Hippocampus ◽  
Regulated Gene Expression ◽  
And Function

ABSTRACT Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3′ untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin α1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.

scholarly journals Pan-cancer analysis of mRNA stability for decoding tumour post-transcriptional programs

2021 ◽  
Author(s):  
Gabrielle Perron ◽  
Pouria Jandaghi ◽  
Maryam Rajaee ◽  
Rached Alkallas ◽  
Yasser Riazalhosseini ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Rna Stability ◽  
Cancer Type ◽  
Multiple Cancers ◽  
Transcriptional Changes ◽  
Potential Factors ◽  
Pan Cancer

AbstractRNA stability is a crucial and often overlooked determinant of gene expression. Some of the regulators of mRNA stability are long known as key oncogenic or tumour suppressor factors. Nonetheless, the extent to which mRNA stability contributes to transcriptome remodeling in cancer is unknown, and the factors that modulate mRNA stability during cancer development and progression are largely uncharacterized. Here, by decoupling transcriptional and post-transcriptional effects in RNA-seq data of 7760 samples from 18 cancer types, we present a pan-cancer view of the mRNA stability changes that accompany tumour development and progression. We show that thousands of genes are dysregulated at the mRNA stability level, and identify the potential factors that drive these changes, including >80 RNA-binding proteins (RBPs) and microRNAs (miRNAs). Most RBPs and miRNAs have cancer type-specific activities, but a few show recurrent inactivation across multiple cancers, including the RBFOX family of RBPs and miR-29. Analysis of cell lines with phenotypic activation or inhibition of RBFOX1 and miR-29 confirms their role in modulation of genes that are dysregulated across multiple cancers, with functions in calcium signaling, extracellular matrix organization, and stemness. Overall, our study highlights the critical role of mRNA stability in shaping the tumour transcriptome, with recurrent post-transcriptional changes that are ~30% as frequent as transcriptional events. These results provide a resource for systematic interrogation of cancer-associated stability drivers and pathways.

scholarly journals Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production

2020 ◽  
Vol 19 (6) ◽  
pp. 542-554
Author(s):  
Marzieh Marzbany ◽  
◽  
Mahsa Rasekhian ◽  
Keyword(s):  
Recombinant Protein ◽  
Mammalian Cell ◽  
Cell Cultures ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Encephalomyocarditis Virus ◽  
Dependent Manner ◽  
Mammalian Cell Cultures

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3′-untranslated region (3′UTR) from RNA viral genome to modulate mRNA stability. The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

scholarly journals Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane

2018 ◽  
Vol 92 (10) ◽  
Author(s):  
Ritsuko Koga ◽  
Marie Kubota ◽  
Takao Hashiguchi ◽  
Yusuke Yanagi ◽  
Shinji Ohno
Keyword(s):  
Plasma Membrane ◽  
Measles Virus ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Annexin A2 ◽  
Host Protein ◽  
Host Cells ◽  
M Protein ◽  
Dependent Manner ◽  
Inner Surface

ABSTRACTAnnexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca2+-dependent manner. Annexin A2 (AnxA2), a member of this family, has been implicated in a variety of cellular functions, including the organization of membrane domains, vesicular trafficking, and cell-cell adhesion. AnxA2 generally forms a heterotetrameric complex with a small Ca2+-binding protein, S100A10. Measles virus (MV), a member of the familyParamyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells without affecting its entry and viral RNA production. In MV-infected, AnxA2 knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10 and was colocalized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6 to 10), thereby aiding in MV assembly.IMPORTANCEMV is an important human pathogen, still claiming ∼100,000 lives per year despite the presence of effective vaccines, and it causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV M protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of antiviral agents.

Sign in / Sign up

Export Citation Format

Share Document