A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.

Short 5′UTR enables optimal translation of plant virus tricistronic RNA via leaky scanning

Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many of mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by read-through of upstream ORFs, is the most major regulatory mechanism to translate polycistronic mRNAs. However, the general regulatory factors controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate that significantly shorter 5′ UTR of the most upstream ORF promotes leaky scanning, potentially finetuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Moreover, in plant endogenous mRNAs, we found that shorter UTRs were more frequently observed in uORFs of polycistronic mRNAs. We propose that the promotion of leaky scanning induced by a short 5′ UTR (LISH), together with the Kozak sequence, is a conserved gene regulation mechanism not only in viruses but also in eukaryotes.

A conserved uORF impacts APOBEC3G translation and is essential for translational inhibition by the HIV-1 Vif protein

ABSTRACTThe HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′UTR) of A3G mRNA. Extensive mutagenesis of A3G 5′-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.

Messenger RNAs with large numbers of upstream open reading frames are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum

The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80–90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

Messenger RNAs with large numbers of upstream ORFs are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum

The genome of Plasmodium falciparum has one of the most skewed base pair compositions of any eukaryote, with an AT content of 80-90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 10 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream gene (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole genome data was analyzed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

Dynamic Interaction of Eukaryotic Initiation Factor 4G1 (eIF4G1) with eIF4E and eIF1 Underlies Scanning-Dependent and -Independent Translation

ABSTRACTTranslation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing thetranslationinitiator ofshort 5′untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.