scholarly journals Cap-Independent Translational Enhancement by the 3′ Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1

2003 ◽  
Vol 77 (22) ◽  
pp. 12113-12121 ◽  
Author(s):  
Hiroyuki Mizumoto ◽  
Masahiro Tatsuta ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Red Clover ◽  
Luciferase Reporter ◽  
Base Substitution ◽  
Luciferase Reporter Gene ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Translational Activity ◽  
Independent Translation

ABSTRACT Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3′ ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5′ cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5′ cap and 3′ poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5′ cap- and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3′ untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5′ cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3′ UTR, designated the 3′ translation element of Dianthovirus RNA1 (3′TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and Luteovirus. A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3′TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.

scholarly journals Cap-Independent Translation Mechanism of Red Clover Necrotic Mosaic Virus RNA2 Differs from That of RNA1 and Is Linked to RNA Replication

2006 ◽  
Vol 80 (8) ◽  
pp. 3781-3791 ◽  
Author(s):  
Hiroyuki Mizumoto ◽  
Hiro-oki Iwakawa ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Time Course ◽  
De Novo ◽  
Red Clover ◽  
Rna Replication ◽  
Firefly Luciferase ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Translational Activity ◽  
Independent Translation

ABSTRACT The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5′ end and no poly(A) tail at the 3′ end. The 3′ untranslated region (3′ UTR) of RCNMV RNA1 contains an essential RNA element (3′TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3′TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5′ UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

scholarly journals The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (20) ◽  
pp. 11284-11289 ◽  
Author(s):  
A. Corina Vlot ◽  
John F. Bol
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Alfalfa Mosaic Virus ◽  
Loop Structure ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Virus Rna

ABSTRACT The three genomic RNAs of alfalfa mosaic virus each contain a unique 5′ untranslated region (5′ UTR). Replacement of the 5′ UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5′ stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5′ stem-loop structure is present in RNA 2 but not in RNA 3.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

scholarly journals miR-373-3p inhibits EMT via regulation of TGFβR2 in choriocarcinoma

2019 ◽  
Author(s):  
yanjie lu ◽  
Xiaoru Li ◽  
Yanzhen Zuo ◽  
qian xu ◽  
lei liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Untranslated Region ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Early Metastasis ◽  
Choriocarcinoma Cells ◽  
Jar Cells

Abstract Background: Previous studies have indicated that early metastasis is a major cause of mortality in patients with choriocarcinoma. However, what determines whether early metastasis of choriocarcinoma has occurred is unknown. The emerging role of miRNA in regulating cancer development and progression has been recognized. MiR-373-3p has been shown to play pivotal roles in tumorigenesis and metastasis. However, whether miR-373-3p functions to promote choriocarcinoma metastasis is not clear. The purpose of this study is to determine the function of miR-373-3p in the progression of choriocarcinoma. Methods: In this study, we first compared EMT-related markers, which are inversely correlated with miR-373-3p expression, in trophoblast and choriocarcinoma cell lines. Using PCR and western blot, the upregulation of miR‑373‑3p was observed to inhibit EMT progression. Similarly, gain-and loss-of-function studies revealed that ectopic miR-373-3p overexpression inhibited the metastasis of choriocarcinoma cells. Results: Our results revealed that miR-373-3p functions as an inhibitor in JEG-3 and JAR cells; this is due to its mediation of the TGF-β signalling pathway, which is responsible for EMT. The bioinformatic analysis and dual‑luciferase reporter gene assays were employed to verify that miR‑373‑3p might interact with the 3' untranslated region of TGFβR2 mRNA. Further western blot results showed miR‑373‑3 preversed the increases of TGFβR2 and inhibited EMT. Conclusions: In light of our observations, miR‑373‑3p upregulation partly accounts for TGFβR2 downregulation and leads to a restraint of EMT and metastasis. MiR‑373‑3p may, therefore, serve as a valuable target in potential anticancer strategies to treat choriocarcinoma.

scholarly journals MSH2 Overexpression Due to an Unclassified Variant in 3’-Untranslated Region in a Patient with Colon Cancer

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 167
Author(s):  
Raffaella Liccardo ◽  
Antonio Nolano ◽  
Matilde Lambiase ◽  
Carlo Della Ragione ◽  
Marina De Rosa ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Tumor Development ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Unclassified Variant ◽  
Dna Mismatch ◽  
Functional Assays ◽  
Mmr Genes ◽  
Uncertain Significance ◽  
Low Expression

Background: The loss or low expression of DNA mismatch repair (MMR) genes can result in genomic instability and tumorigenesis. One such gene, MSH2, is mutated or rearranged in Lynch syndrome (LS), which is characterized by a high risk of tumor development, including colorectal cancer. However, many variants identified in this gene are often defined as variants of uncertain significance (VUS). In this study, we selected a variant in the 3′ untranslated region (UTR) of MSH2 (c*226A > G), identified in three affected members of a LS family and already reported in the literature as a VUS. Methods: The effect of this variant on the activity of the MMR complex was examined using a set of functional assays to evaluate MSH2 expression. Results: We found MSH2 was overexpressed compared to healthy controls, as determined by RTqPCR and Western blot analyses of total RNA and proteins, respectively, extracted from peripheral blood samples. These results were confirmed by luciferase reporter gene assays. Conclusions: We therefore speculated that, in addition to canonical inactivation via a gene mutation, MMR activity may also be modulated by changes in MMR gene expression.

scholarly journals Host-dependent roles of the viral 5′ untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3′ UTR of Red clover necrotic mosaic virus RNA1

Virology ◽  
2009 ◽  
Vol 391 (1) ◽  
pp. 107-118 ◽  
Author(s):  
Siriruk Sarawaneeyaruk ◽  
Hiro-oki Iwakawa ◽  
Hiroyuki Mizumoto ◽  
Hiromi Murakami ◽  
Masanori Kaido ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Red Clover ◽  
Rna Stabilization
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