Добірка наукової літератури з теми "Ribosomal leaky scanning"

Abstract eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5′ UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5′ UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

ABSTRACT β-Site β-amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is the β-secretase in vivo for processing APP to generate amyloid β protein (Aβ). Aβ deposition in the brain is the hallmark of Alzheimer's disease (AD) neuropathology. Inhibition of BACE1 activity has major pharmaceutical potential for AD treatment. The expression of the BACE1 gene is relatively low in vivo. The control of BACE1 expression has not been well defined. There are six upstream AUGs (uAUGs) in the 5′ leader sequence of the human BACE1 mRNA. We investigated the role of the promoter and the uATGs in the 5′ untranslated region (UTR) of the human BACE1 gene in BACE1 gene transcription and translation initiation. Our results show that the first and second uATGs are the integral part of the core minimal promoter of the human BACE1 gene, while the third uAUG is skipped over by ribosomal scanning. The fourth uAUG can function as a translation initiation codon, and deletion or mutation of this uAUG increases downstream gene expression. The fourth uAUG of the BACE1 5′UTR is responsible for inhibiting the expression of BACE1. Translation initiation by the BACE1 uAUGs and physiological AUG requires intact eIF4G. Our results demonstrate that during human BACE1 gene expression, ribosomes skipped some uAUGs by leaky scanning and translated an upstream open reading frame, initiated efficiently at the fourth uAUG, and subsequently reinitiated BACE1 translation at the physiological AUG site. Such leaky scanning and reinitiation resulted in weak expression of BACE1 under normal conditions. Alterations of the leaky scanning and reinitiation in BACE1 gene expression could play an important role in AD pathogenesis.

Abstract During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.

To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

ABSTRACT Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

ABSTRACT The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C′, C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C′ to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5′ cap and then scan ca. 50 nucleotides (nt) of the 5′ untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

Le virus Nipah (NiV) est un virus à ARN à polarité négative non segmenté appartenant à la famille des Paramyxoviridae. Le génome de NiV ne comporte que 6 gènes, mais code pour 9 protéines différentes. Plusieurs protéines sont exprimées à partir du gène P dont la phosphoprotéine P et la protéine C. La protéine C est exprimée à partir d'un cadre de lecture ouvert alternatif par un mécanisme du balayage fuyant ribosomique. La protéine C joue un rôle encore mal compris dans la réplication virale. Pour étudier sa fonction, de nombreuses recherches ont été réalisées en caractérisant un virus recombinant déficient dans l’expression de C (rNiV CKO) où les codons initiateurs de C ont été supprimés, sans toutefois altérer les autres cadres de lecture des protéines issues du gène P. Nous avons émis l’hypothèse que l’absence du site d’initiation de la traduction de C n’empêche pas le mécanisme de balayage ribosomal déficient mais que ces mutations entraînent une désorganisation de l’expression des protéines exprimées à partir du gène P. Nous avons confirmé cette hypothèse en démontrant que le virus rNiV CKO, exprime des formes tronquées des protéines C et P, nommées C’ et P’ respectivement. Par ailleurs, notre évaluation comparative des virus rNiV CKO et NiV sauvage (WT) a révélé que le rNiV CKO produit des particules virales moins infectieuses supposant ainsi un impact des protéines C, C’ et P’ sur la réplication virale. Nous avons développé un système de minigénome NiV et étudié l’effet de ces protéines sur l’activité de la polymérase virale de ce système. C et C' ont montré une régulation négative de l'activité de la polymérase, avec une régulation plus marquée par C'. De plus, nous avons observé des différences de localisation cellulaire entre C et C', cette dernière se localisant dans les corps d'inclusion en présence de protéines virales impliquées dans la synthèse de l'ARN. Concernant la protéine P’, bien qu'elle ait perdu sa fonction principale de liaison aux nucléoprotéines monomériques, aucun effet dominant négatif n'a été observé sur l'activité de la polymérase NiV. De surcroît, un mélange de P’ et de P dépourvues de leur domaine C-terminal Px peut efficacement substituer la P sauvage dans le système de minigénome, offrant ainsi de nouvelles clés de compréhension sur le mécanisme de réplication du NiV. En conclusion, nos résultats suggèrent que l'organisation de l'expression du gène P est complexe, notamment en raison de deux exigences interdépendantes : la synthèse de C dépend d'une initiation faible de la traduction de P, et l’initiation de la traduction de la protéine C est nécessaire pour prévenir une expression potentiellement aberrante de formes tronquées de C et de P
The Nipah virus (NiV) is a non-segmented negative-sense RNA virus belonging to the Paramyxoviridae family. The NiV genome contains only 6 genes but codes for 9 different proteins. Several proteins are expressed from the P gene, including the phosphoprotein P and the C protein. The C protein, expressed from an alternative open reading frame through a ribosomal leaky scanning mechanism, plays a yet poorly understood role in viral replication. To study its function, numerous studies have been conducted on a recombinant NiV deficient in C expression (rNiV CKO), where the initiator codons of C have been removed, without altering other reading frames of the proteins derived from the P gene. We hypothesized that the absence of the C translation initiation site does not prevent the ribosomal leaky scanning mechanism but that these mutations lead to a disorganization of the expression of proteins derived from the P gene. We confirmed this hypothesis by demonstrating that rNiV CKO expresses truncated forms of the C and P proteins, named respectively C’ and P’. Furthermore, our comparative evaluation of rNiV CKO and wild-type NiV (WT) revealed that rNiV CKO produces less infectious viral particles, which directed our research towards the impact of C, C', and P' proteins on viral replication. We developed a NiV minigenome system and studied the effect of these proteins on the viral polymerase activity in this system. C and C' showed negative regulation of polymerase activity, with a more pronounced regulation by C'. Additionally, we observed differences in cellular localization between C and C', with the latter localizing in inclusion bodies in the presence of viral proteins involved in RNA synthesis. Regarding the P' protein, although it lost its primary function of binding to monomeric nucleoproteins, no dominant negative effect was observed on NiV polymerase activity. Moreover, a mixture of P’ and P lacking their C-terminal Px domain can effectively substitute for wild-type P in the minigenome system, providing new insights into the NiV replication mechanism. In conclusion, our results collectively suggest that the organization of P gene expression is complex, likely due to two interdependent requirements: the synthesis of C depends on weak initiation of P translation, and the translation initiation of C protein is necessary to prevent potentially aberrant expression of truncated forms of C and P