scholarly journals A novel role for poly(C) binding proteins in programmed ribosomal frameshifting

2016 ◽  
Vol 44 (12) ◽  
pp. 5491-5503 ◽  
Author(s):  
Sawsan Napthine ◽  
Emmely E. Treffers ◽  
Susanne Bell ◽  
Ian Goodfellow ◽  
Ying Fang ◽  
...  
Keyword(s):  
Translational Control ◽  
Binding Proteins ◽  
Cellular Protein ◽  
In Vitro Translation ◽  
Respiratory Syndrome Virus ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Protein Factor ◽  
Slippery Sequence

Abstract Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

scholarly journals Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus

2019 ◽  
Vol 47 (15) ◽  
pp. 8207-8223 ◽  
Author(s):  
Sawsan Napthine ◽  
Susanne Bell ◽  
Chris H Hill ◽  
Ian Brierley ◽  
Andrew E Firth
Keyword(s):  
Virus Infection ◽  
Rna Structure ◽  
Encephalomyocarditis Virus ◽  
In Vitro Translation ◽  
Theiler's Murine Encephalomyelitis Virus ◽  
Ribosomal Frameshifting ◽  
Theiler’S Murine Encephalomyelitis Virus ◽  
Encephalomyelitis Virus

AbstractMany viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

scholarly journals Delayed Translational Silencing of Ceruloplasmin Transcript in Gamma Interferon-Activated U937 Monocytic Cells: Role of the 3′ Untranslated Region

1999 ◽  
Vol 19 (10) ◽  
pp. 6898-6905 ◽  
Author(s):  
Barsanjit Mazumder ◽  
Paul L. Fox
Keyword(s):  
Translational Control ◽  
Untranslated Region ◽  
Amine Oxidase ◽  
Gamma Interferon ◽  
In Vitro Translation ◽  
Deletion Mapping ◽  
Monocytic Cells ◽  
Cytokine Activation ◽  
Ifn Γ

ABSTRACT Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-γ) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-γ for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-γ treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-γ for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3′ untranslated region (3′-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3′-UTR added as a “decoy” and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3′-UTR indicated an internal 100-nucleotide region of the Cp 3′-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

scholarly journals Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control

1992 ◽  
Vol 164 (1) ◽  
pp. 103-116 ◽  
Author(s):  
G. E. Hofmann ◽  
S. C. Hand
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Messenger Rna ◽  
Artemia Franciscana ◽  
In Vitro Translation ◽  
Translation Techniques ◽  
Polysome Profile ◽  
Anaerobic Dormancy ◽  
Qualitative Changes

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.

scholarly journals A memory of eS25 loss drives resistance phenotypes

2020 ◽  
Author(s):  
Alex G Johnson ◽  
Ryan A Flynn ◽  
Christopher P Lapointe ◽  
Yaw Shin Ooi ◽  
Michael L Zhao ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Ribosomal Protein Gene ◽  
Human Cell Line ◽  
Cellular Protein ◽  
Ribosomal Protein Genes ◽  
Genomic Locus ◽  
Cellular Phenotypes

Abstract In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

scholarly journals 2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 106 ◽  
Author(s):  
Piero Monaco ◽  
Virginie Marcel ◽  
Jean-Jacques Diaz ◽  
Frédéric Catez
Keyword(s):  
Ribosomal Rna ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
In Vitro Translation ◽  
Ribosome Structure ◽  
Quantitative Mapping ◽  
And Function ◽  
The Impact ◽  
First Time

Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.

In vitro translation of mRNAs that are in their native ribonucleoprotein complexes

2015 ◽  
Vol 472 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Baptiste Panthu ◽  
Fabrice Mure ◽  
Henri Gruffat ◽  
Theophile Ohlmann
Keyword(s):  
Binding Proteins ◽  
In Vitro Translation ◽  
Ribosome Binding ◽  
Ribonucleoprotein Complexes ◽  
The Impact

The present study shows the impact of ribosome binding proteins on in vitro translation.

scholarly journals Novel Translational Control through an Iron-Responsive Element by Interaction of Multifunctional Protein YB-1 and IRP2

2002 ◽  
Vol 22 (18) ◽  
pp. 6375-6383 ◽  
Author(s):  
Megumi Ashizuka ◽  
Takao Fukuda ◽  
Takanori Nakamura ◽  
Kanemitsu Shirasuna ◽  
Kazuhiro Iwai ◽  
...  
Keyword(s):  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
In Vitro Translation ◽  
High Concentration ◽  
Iron Responsive Element ◽  
Responsive Element

ABSTRACT The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5′ untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5′UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.

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