A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

ABSTRACT Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5′ element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5′ and 3′ elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3′ enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3′ splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

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A Premature Termination Codon in Either Exon of Minute Virus of Mice P4 Promoter-generated Pre-mRNA Can Inhibit Nuclear Splicing of the Intervening Intron in an Open Reading Frame-dependent Manner

Journal of Biological Chemistry ◽

10.1074/jbc.274.32.22452 ◽

1999 ◽

Vol 274 (32) ◽

pp. 22452-22458 ◽

Cited By ~ 20

Author(s):

Anand Gersappe ◽

Lisa Burger ◽

David J. Pintel

Keyword(s):

Premature Termination ◽

Premature Termination Codon ◽

Open Reading Frame ◽

Termination Codon ◽

Dependent Manner ◽

Minute Virus Of Mice ◽

Reading Frame ◽

Minute Virus

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Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8562-8570.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8562-8570 ◽

Cited By ~ 48

Author(s):

Deanna M. Janzen ◽

Lyudmila Frolova ◽

Adam P. Geballe

Keyword(s):

Translation Termination ◽

Open Reading Frame ◽

Upstream Open Reading Frame ◽

Dependent Manner ◽

Release Factor ◽

Inhibitory Mechanism ◽

Reading Frame ◽

Nascent Peptide ◽

Peptide Product

ABSTRACT Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.

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Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1986 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1986-1996 ◽

Cited By ~ 21

Author(s):

G F Barker ◽

K Beemon

Keyword(s):

Rous Sarcoma Virus ◽

Rna Stability ◽

Subcellular Fractionation ◽

Open Reading Frame ◽

Termination Codon ◽

Gag Protein ◽

Reading Frame ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Nonsense Codons

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

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Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1986-1996.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1986-1996

Author(s):

G F Barker ◽

K Beemon

Keyword(s):

Rous Sarcoma Virus ◽

Rna Stability ◽

Subcellular Fractionation ◽

Open Reading Frame ◽

Termination Codon ◽

Gag Protein ◽

Reading Frame ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Nonsense Codons

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Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

Journal of Virology ◽

10.1128/jvi.02113-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2119-2129 ◽

Cited By ~ 23

Author(s):

Jason E. Weil ◽

Michalis Hadjithomas ◽

Karen L. Beemon

Keyword(s):

Secondary Structure ◽

Rous Sarcoma Virus ◽

Translation Termination ◽

Rna Stability ◽

Premature Termination Codon ◽

Termination Codon ◽

Stem Loop ◽

Future Design ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3′ untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3′ UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5′ end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

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Identification of a Pneumococcal Glycosidase That Modifies O-Linked Glycans

Infection and Immunity ◽

10.1128/iai.01215-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1389-1396 ◽

Cited By ~ 40

Author(s):

Carolyn Marion ◽

Dominique H. Limoli ◽

Gregory S. Bobulsky ◽

Jessica L. Abraham ◽

Amanda M. Burnaugh ◽

...

Keyword(s):

Sequence Similarity ◽

Bifidobacterium Longum ◽

Airway Epithelial Cells ◽

Open Reading Frame ◽

Glycan Structure ◽

Dependent Manner ◽

Upper Respiratory Tract ◽

Reading Frame ◽

Human Airway ◽

Published Evidence

ABSTRACT Colonization of the airway by Streptococcus pneumoniae is typically asymptomatic; however, progression of bacteria beyond the oronasopharynx can cause diseases including otitis media and pneumonia. The mechanisms by which S. pneumoniae establishes and maintains colonization remain poorly understood. Both N-linked and O-linked glycans are abundant in the airway. Our previous research demonstrated that S. pneumoniae can sequentially deglycosylate N-linked glycans and suggested that this modification of sugar structures may aid in colonization. There is published evidence that S. pneumoniae expresses a secreted O-glycosidase that cleaves galactose β1-3 N-acetylgalactosamine (Galβ1-3GalNAc) from core-1 O-linked glycans; however, the biological function of this enzyme has not previously been determined. We established that the activity is not secreted but is instead surface associated in a sortase-dependent manner. Genome analysis revealed an open reading frame predicted to encode a sortase-dependent surface protein with sequence similarity to the O-glycosidase of Bifidobacterium longum. Deletion of this pneumococcal open reading frame confirmed that this gene encodes an O-glycosidase. Experiments using a model glycoconjugate demonstrated that this O-glycosidase, together with the neuraminidase NanA, is required for S. pneumoniae to cleave sialylated core-1 O-linked glycans. The ability of the O-glycosidase mutant to cleave this glycan structure was restored by both genetic complementation and the addition of O-glycosidase. The mutant showed a reduction in adherence to human airway epithelial cells and a significantly decreased ability to colonize the upper respiratory tract, suggesting that cleavage of core-1 O-linked glycans enhances the ability of S. pneumoniae to colonize the human airway.

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Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA

Journal of Virology ◽

10.1128/jvi.02362-07 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8917-8921 ◽

Cited By ~ 21

Author(s):

Christopher J. McCormick ◽

Omar Salim ◽

Paul R. Lambden ◽

Ian N. Clarke

Keyword(s):

Hepg2 Cells ◽

Nonstructural Protein ◽

Translation Termination ◽

Surrogate Marker ◽

Open Reading Frames ◽

Open Reading Frame ◽

Subgenomic Rna ◽

Reading Frame ◽

Reading Frames ◽

Open Reading Frame 1

ABSTRACT A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.

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Faculty Opinions recommendation of Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018386.208429 ◽

2004 ◽

Author(s):

Javier Paz-Ares

Keyword(s):

Medicago Truncatula ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Open Reading Frame ◽

Cytoplasmic Localization ◽

Reading Frame ◽

Short Open Reading Frame ◽

Nuclear Rna

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Molecular Mechanism of Prothrombin G20210A Variant: Critical New Role of Exon Splicing Enhancer in Poly (A) Tailing.

Blood ◽

10.1182/blood.v104.11.1944.1944 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1944-1944 ◽

Cited By ~ 2

Author(s):

Mitsuhiro Kuwahara ◽

Sumiko Kurachi ◽

Kotoku Kurachi

Keyword(s):

Molecular Mechanism ◽

Mrna Level ◽

Critical Role ◽

Rna Stability ◽

Prothrombin G20210a ◽

Coding Regions ◽

Culture Cells ◽

Exon Splicing ◽

Prothrombin Gene ◽

Splicing Enhancer

Abstract Although prothrombin G20210A variant was first found in 1996 as a genetic risk factor of thrombosis, the underlying molecular mechanism for elevation of circulatory prothrombin levels due to the variant has remained elusive. The position 20210 is at the poly (A) tailing site, and several research groups reported different, contradictive possibilities including enhancement in poly (A) tailing, change in RNA stability or no difference in prothrombin mRNA level between G (wild type) and A (variant) at position 20210. Such confusions appear to be due to no reliable assay model availability. To overcome these problems, we developed a minigene assay system composed of only autologous components of human prothrombin gene, including its promoter, coding regions, introns and 3′UTR with poly (A) signal. A minigene containing the first intron and the last intron did not express at any appreciable levels, failing proper splicing of the last intron. Inclusion of upstream other introns was found essential for its appropriate splicing. Although it is generally accepted that the last intron enhances the efficiency of poly (A) tailing, we found that it is not the case for the prothrombin gene. Instead, an exon-splicing enhancer (ESE) of another upstream intron was found to be responsible for efficient poly (A) tailing. ESE, therefore, plays a critical role not only in alternative splicing but also in constitutive RNA processing, including poly (A) tailing. Etiology of prothrombin G20210A variant studied with culture cells and transgenic mice, supported an enhancement of poly (A) tailing, and not a change in mRNA stability.

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