Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.     

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.     

Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons

The use of several translation initiation codons in a single mRNA, by expressing several proteins from a single gene, contributes to the generation of protein diversity. A small, yet growing, number of mammalian mRNAs initiate translation from a non-AUG codon, in addition to initiating at a downstream in-frame AUG codon. Translation initiation on such mRNAs results in the synthesis of proteins harbouring different amino terminal domains potentially conferring on these isoforms distinct functions. Use of non-AUG codons appears to be governed by several features, including the sequence context and the secondary structure surrounding the codon. Selection of the downstream initiation codon can occur by leaky scanning of the 43S ribosomal subunit, internal entry of ribosome or ribosomal shunting. The biological significance of non-AUG alternative initiation is demonstrated by the different subcellular localisations and/or distinct biological functions of the isoforms translated from the single mRNA as illustrated by the two main angiogenic factor genes encoding the fibroblast growth factor 2 (FGF2) and the vascular endothelial growth factor (VEGF). Consequently, the regulation of alternative initiation of translation might have a crucial role for the biological function of the gene product.     

Translational polymorphism as a potential source of plant proteins variety in Arabidopsis thaliana

MOTIVATION: According to scanning model, 40S ribosomal subunits can either initiate translation at start AUG codon in suboptimal context or miss it and initiate translation at downstream AUG(s), thereby producing several proteins. Functional significance of such a protein translational polymorphism is still unknown. RESULTS: We compared predicted subcellular localizations of annotated Arabidopsis thaliana proteins and their potential N-terminally truncated forms started from the nearest downstream in-frame AUG codons. It was found that localizations of full and N-truncated proteins differ in many cases: 12.2% of N-truncated proteins acquired sorting signals de novo and 5.7% changed their predicted subcellular locations (mitochodria, chloroplast or secretory pathway). It is likely that the in-frame downstream AUGs may be frequently utilized to synthesize proteins possessing new functional properties and such a translational polymorphism may serve as an important source of cellular and organelle proteomes.     

5’UTR中AUG的分布及其对翻译起始的影响

以细菌和古菌基因组5′ UTR序列作为研究对象,分析在5′ UTR 的3个不同阅读框架中三联体AUG的分布,发现无论是细菌还是古菌基因组都在阅读框1中有非常明显的AUG缺失(depletion)。AUG的缺失表明在起始密码子上游的AUG很可能会对基因的翻译起始产生影响。分析得知：绝大部分的AUG都是以uORF(upstream open reading frame)的形式出现的,uAUG(upstream AUG)的数量很少,特别是在阅读框1中,而且在细菌基因组的阅读框1中uAUG较多地出现在了含有SD序列的基因上游。比较发现,uAUG引导的序列在同义密码子使用上的偏好性较真正的编码序列差,这可能表明细菌和古菌在同义密码子使用上的偏好性也是决定基因准确地翻译起始的重要因素之一。     

Overlapping signals for translational regulation and packaging of influenza A virus segment 2

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.     

Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2

The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.     

Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis.     

Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.     

Identification of a cis-acting element required for shunt-mediated translational initiation of the Sendai virus Y proteins

Shunting is a mechanism that permits translational initiation at internal codons positioned in proximity to a ribosome acceptor sequence. Sendai virus exploits shunting to express a series of proteins that initiate at the fourth and fifth start sites on the P/C mRNA (namely, the Y1 and Y2 proteins, respectively). Shunt-mediated initiation at these sites is codon independent. In an attempt to characterise the acceptor site, an extensive deletion analysis was performed spanning the entire C ORF. Only mutants flanking the Y1/Y2 start sites exhibited altered shunt phenotypes. Some of these significantly enhanced shunting efficiency to the point where the Y1/Y2 proteins were the major translational products of the mRNA. Additionally, removal of a short region just downstream of the Y2 start codon (referred to as Δ10) ablated all Y protein initiation via shunting but had no effect on Y expression when the AUG codons were viewed by a scanning ribosome. Point mutations introduced into this Δ10 sequence severely perturbed shunt-mediated initiation. We also provide evidence that changes in this region of the P/C mRNA may be used to modulate Y protein expression levels in different viral strains.     

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.     

The structure of the 5'-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5'-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG(356). Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG(191), leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG(191) and AUG(356) inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5'-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation.     

Identification of two separable modules in the duck hepatitis B virus core protein.

Hepadnavirus replication requires the concerted action of the polymerase and core proteins to ensure packaging of the RNA pregenome and DNA maturation. The arginine-rich C terminus of the core protein plays an essential role in both of these steps while being dispensable for nucleocapsid formation. In an attempt to identify other functional domains of the core protein, we performed a series of trans-complementation experiments analyzing the ability of duck and human hepatitis B virus (DHBV and HBV) core protein subunits to support the replication of a core-defective DHBV genome. Plasmids expressing the N-terminal amino acids 1 to 67 or the remaining C-terminal portion, amino acids 67 to 262, of the DHBV core protein were cotransfected into LMH cells along with a replication-deficient construct coding for the DHBV pregenome and polymerase. Neither the N nor the C terminus alone yielded replication-competent core particles. However, cotransfection of plasmids that separately expressed both regions restored a normal replication pattern. Furthermore, the DHBV C terminus but not the N terminus could be replaced by the corresponding domain of the HBV core protein in this assay. Finally, coexpression of the complete HBV core protein and the N terminus from DHBV resulted in DHBV replication, while the HBV core protein alone was not functional. Taken together, these findings suggest a modular organization of the DHBV core protein in which the C terminus is functionally conserved among different hepadnaviruses.