Phosphorylation of eIF2α is required for mRNA translation inhibition and survival during moderate hypoxia

ABSTRACT Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

Download Full-text

Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition

eLife ◽

10.7554/elife.66369 ◽

2021 ◽

Vol 10 ◽

Author(s):

Fatima Alghoul ◽

Schaeffer Laure ◽

Gilbert Eriani ◽

Franck Martin

Keyword(s):

Molecular Mechanisms ◽

Stop Codon ◽

Mrna Translation ◽

Upstream Open Reading Frame ◽

Initiation Factor ◽

Entry Site ◽

80S Ribosome ◽

Stem Loop ◽

Reading Frame ◽

Translation Inhibition

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The ‘start-stop’ sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

Download Full-text

Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

10.1101/2021.01.19.427285 ◽

2021 ◽

Author(s):

Fatima Alghoul ◽

Laure Schaeffer ◽

Gilbert Eriani ◽

Franck Martin

Keyword(s):

Molecular Mechanisms ◽

Stop Codon ◽

Mrna Translation ◽

Upstream Open Reading Frame ◽

Initiation Factor ◽

Entry Site ◽

80S Ribosome ◽

Stem Loop ◽

Reading Frame ◽

Translation Inhibition

AbstractDuring embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The a11 ‘start-stop’ sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

Download Full-text

The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through its RGG and C-terminal domains

10.1101/2020.06.25.171967 ◽

2020 ◽

Author(s):

Youssi M. Athar ◽

Simpson Joseph

Keyword(s):

Mental Retardation ◽

Rna Binding ◽

Fragile X ◽

Mrna Translation ◽

Translation Inhibition ◽

Fragile X Mental Retardation ◽

Mental Retardation Protein ◽

Elongation Step ◽

Independent Translation

AbstractFragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates the translation of numerous mRNAs in neurons. The precise mechanism of translational regulation by FMRP is unknown. Some studies have indicated that FMRP inhibits the initiation step of translation, whereas other studies have indicated that the elongation step of translation is inhibited by FMRP. To determine whether FMRP inhibits the initiation or the elongation step of protein synthesis, we investigated m7G-cap-dependent and IRES-driven, cap-independent translation of several reporter mRNAs in vitro. Our results show that FMRP inhibits both m7G-cap-dependent and cap-independent translation to similar degrees, indicating that the elongation step of translation is inhibited by FMRP. Additionally, we dissected the RNA-binding domains of hFMRP to determine the essential domains for inhibiting translation. We show that the RGG domain, together with the C-terminal domain (CTD), is sufficient to inhibit translation while the KH domains do not inhibit mRNA translation. However, the region between the RGG domain and the KH2 domain may contribute as NT-hFMRP shows more potent inhibition than the RGG-CTD tail alone. Interestingly, we see a correlation between ribosome binding and translation inhibition, suggesting the RGG-CTD tail of hFMRP may anchor FMRP to the ribosome during translation inhibition.

Download Full-text

The different activities of RNA G-quadruplex structures are controlled by flanking sequences

Life Science Alliance ◽

10.26508/lsa.202101232 ◽

2021 ◽

Vol 5 (2) ◽

pp. e202101232

Author(s):

Alice J-L Zheng ◽

Aikaterini Thermou ◽

Pedro Guixens Gallardo ◽

Laurence Malbert-Colas ◽

Chrysoula Daskalogianni ◽

...

Keyword(s):

Immune Evasion ◽

Mrna Translation ◽

Repeat Sequence ◽

Rna Structures ◽

Coding Sequences ◽

Translation Inhibition ◽

G Quadruplex ◽

Flanking Sequences ◽

Context Dependent

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

Download Full-text

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2105274118 ◽

2021 ◽

Vol 118 (35) ◽

pp. e2105274118

Author(s):

Chuande Wang ◽

Lina Lezhneva ◽

Nadège Arnal ◽

Martine Quadrado ◽

Hakim Mireau

Keyword(s):

Messenger Rna ◽

Mrna Translation ◽

Pollen Sterility ◽

Genetic Interactions ◽

Gene Control ◽

Fertility Restorer ◽

Translation Elongation ◽

Translation Inhibition ◽

Restorer Of Fertility ◽

Fertility Restorers

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

Download Full-text

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00602.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. G1025-G1032 ◽

Cited By ~ 43

Author(s):

Dharmalingam Subramaniam ◽

Gopalan Natarajan ◽

Satish Ramalingam ◽

Ilangovan Ramachandran ◽

Randal May ◽

...

Keyword(s):

Cell Cycle ◽

Colon Cancer ◽

Cancer Cells ◽

Rna Binding ◽

Mrna Translation ◽

Cyclin B1 ◽

Colon Cancer Cells ◽

Translation Inhibition ◽

M Phase ◽

Hct 116

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G2-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for α-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G2-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3′-untranslated region (3′-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3′-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3′-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G2 phase of the cell cycle.

Download Full-text