Ribosome Scanning Mechanism Research Articles

Computational analysis and mapping of novel open reading frames in influenza A viruses.

The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11th viral protein, PB1-F2, which uses an alternative reading frame of the PB1 gene. The recently identified PB1-N40, PA-N155 and PA-N182 influenza A proteins have shown the potential for using a leaking ribosomal scanning mechanism to generate novel open reading frames (ORFs). These novel ORFs provide examples of the manner in which the influenza A virus expands its coding capacity by using overlapping reading frames. In this study, we performed a computational search, based on a ribosome scanning mechanism, on all influenza A coding sequences to identify possible forward-reading ORFs that could be translated into novel viral proteins. We specified that the translated products had a prevalence ≥5% to eliminate sporadic ORFs. A total of 1,982 ORFs were thus identified and presented in terms of their locations, lengths and Kozak sequence strengths. We further provided an abridged list of ORFs by requiring every candidate an upstream start codon (within the upstream third of the primary transcript), a strong Kozak consensus sequence and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively). The PB1-F2, PB1-N40, PA-N155 and PA-N182 proteins all fulfilled our filtering criteria. Subject to these three stringent settings, we additionally named 16 novel ORFs for all influenza A genomes except for HA and NA, for which 43 HA and 11 NA ORFs from their respective subtypes were also recognized.

Different Mechanisms Preserve Translation of Programmed Cell Death 8 and JunB in Virus-Infected Endothelial Cells

Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown. We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription-polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5' untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.

Host-dependent roles of the viral 5′ untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3′ UTR of Red clover necrotic mosaic virus RNA1

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3'TE-DR1) in the 3' untranslated region (UTR). In this study, we analyzed the roles of 5' and 3' UTRs of RNA1 in 3'TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (Luc) reporter assay system. Most mutations introduced into RNA1 5' UTR in reporter Luc mRNA abolished or greatly reduced cap-independent translation in BY-2 protoplasts, whereas those mutations had no or much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stem-loop structure predicted in the 5' proximal region of RNA1 plays important roles in both translation and RNA stability. We also show that 3'TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts.

A minimal uORF within the HIV-1 vpu leader allows efficient translation initiation at the downstream env AUG

The HIV-1 Vpu and Env proteins are translated from 16 alternatively spliced bicistronic mRNA isoforms. Translation of HIV-1 mRNAs generally follows the ribosome scanning mechanism. However, by using subgenomic env expression vectors, we found that translation of glycoprotein from polycistronic mRNAs was inconsistent with leaky scanning. Instead a conserved minimal upstream open reading frame (uORF) consisting only of a start and stop codon that overlaps with the vpu start site, appears to augment access to the env start codon downstream. Mutating the translational start and stop codons of this uORF resulted in up to fivefold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter Env expression.

Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs

Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5' untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5' UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.

An evolutionarily conserved translation initiation mechanism regulates nuclear or mitochondrial targeting of DNA ligase 1 in Arabidopsis thaliana

The Arabidopsis DNA ligase 1 gene (AtLIG1) is indispensable for cell viability. AtLIG1 expresses one major and two minor mRNA transcripts differing only in the length of the 5' untranslated leader sequences preceding a common ORF. Control of AtLIG1 isoform production and intracellular targeting depends upon mechanisms controlling the choice of translation initiation site within the AtLIG1 ORF. Confocal laser scanning microscopy of green fluorescent protein-tagged AtLIG1 isoforms expressed in Arabidopsis revealed that translation of AtLIG1 mRNA transcripts from the first in-frame start codon produces an AtLIG1 isoform that is targeted exclusively to the mitochondria. Translation initiation from the second in-frame start codon produces an AtLIG1 isoform targeted only to the nucleus. There is no evidence for AtLIG1-GFP being targeted to chloroplasts. The mitochondrial AtLIG1 isoform possesses both an N-terminal mitochondrial-targeting signal and an internal bipartite nuclear localization signal (NLS) yet is targeted only to mitochondria, demonstrating a hierarchical dominance of the mitochondrial presequence over the NLS. The length of the 5'-UTR and more significantly the nucleotide context around alternative start codons in the AtLIG1 transcripts affect translation initiation to ensure a balanced synthesis of both nuclear and mitochondrial AtLIG1 isoforms, probably via a context-dependent leaky ribosome scanning mechanism.

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.

Structural characterization of the gene encoding the large zinc finger protein ZAS3: implication to the origin of multiple promoters in eukaryotic genes

ZAS3 is a large zinc finger protein that regulates κB-mediated transcription and TNF-driven signal transduction pathway. Herein, we have characterized the mouse ZAS3 gene that spans 400 kb and splits into 16 exons. Four ZAS3 exons, ranging from 676 to 3956 nucleotides, are significantly larger than the average size of mammalian internal exons. Intron 10, when retained in mRNAs, encodes N-terminal DNA binding domain, called ZASN. As predicted from cDNAs, 5′ untranslated region composed of the 2317 nucleotides is extremely long and contains upstream open reading frames, suggesting that translation initiation of ZAS3 transcripts by conventional cap-dependent ribosome scanning mechanism may be inefficient. Additionally, cDNA data analysis followed by reporter gene assays shows that the ZAS3 locus harbors two promoters that are 80 kb apart. The data suggest that the expression of ZAS3 is controlled by a combination of differential promoter usage, alternative splicing, and possible intergenic splicing. The distribution and degree of conservation of exons within the ZAS3 locus, together with the complex alternative splicing events and upstream open reading frame in 5′ untranslated exons, lead us to speculate that multiple promoters of an eukaryotic gene might be residual traces of regulatory regions of other genes lost in evolution.

Regulation of CCAAT/enhancer-binding protein-beta isoform synthesis by alternative translational initiation at multiple AUG start sites.

The mRNA of the intronless, single-copy CCAAT/enhancer-binding protein-beta (C/EBPbeta) gene encodes several isoforms that have truncated transcription activation domains. This occurs by the alternative translational initiation (ATI) at multiple AUG start sites. The C/EBPbeta mRNA has four in-frame AUGs and an internal out-of-frame AUG associated with a small open reading frame (sORF). Initiation of translation at the in-frame AUGs forms 40-kDa (AUG-1), 35-kDa (AUG-2), 20-kDa (AUG-3) and 8.5-kDa (AUG-4) isoforms. We show that in COS-1 cells the 20-kDa isoform is not a product of proteolysis of the higher molecular weight isoforms. The sORF contains an AUG and termination signal that may produce the oligopeptide MPPAAARRL. Our studies suggest that ATI involves three mRNA structural features: (i) the cap structure, (ii) the context of the Kozak sequences that flank the AUG and (iii) the integrity of the sORF. We propose that formation of C/EBPbeta isoforms is accomplished by a leaky ribosomal scanning mechanism that facilitates ATI of multiple internal AUGs.

Identification of a noncanonical transcription initiation site for transcription of a subgenomic mRNA of mouse hepatitis virus.

Subgenomic mRNA transcription of mouse hepatitis virus (MHV) involves the interaction between the leader and the intergenic (IG) sequence of the template. All mRNAs contain multiple open reading frames (ORF) with the exception of the smallest mRNA. In general, only the 5’-most ORF of each mRNA is translated into a protein via the cap-dependent ribosomal scanning mechanism, while the down-stream ORFs are not translatable. The E (envelope) protein of MHV is a structural protein (Yu et al., 1994), essential for virion assembly. It is translated from the downstream ORF of the bicistronic mRNA5 in vitro (Thiel and Siddell, 1995), suggesting that translation of the E protein is cap-independent, possibly via an internal ribosomal entry site (IRES). Subsequently, the IRES has been mapped through a series of deletion mutants in an in vitro translation assay to be located between ≈100-nt upstream and 180-nt downstream of the ORF5b (Jendrach et al., 1999). The gene encoding the E in other coronaviruses includes ORF5b in bovine Coronavirus (BCoV), ORF4 in transmissible gastroenteritis virus (TGEV) and human Coronavirus (HCoV) 229E, and ORF3c in avian infectious bronchititis virus (IBV). Interestingly, while the ORF4 of TGEV and HCoV-229E, and ORF5b of BCoV are translated via the cap-dependent mechanism from a distinct mRNA species, mRNA4 and mRNAS-1, respectively, the 0RF3C of IBV and ORF5b of MHV are translated via an IRES from a downstream ORF of mRNA3 and mRNA5, respectively (see review by Lai and Cavanagh, 1997). More intriguingly, within TGEV strains, the ORF3b of Muller strain is translated from mRNA3 via internal ribosomal entry whereas that of the Purdue strain is translated from mRNA3-l, an mRNA species differing from mRNA3, via cap-dependent mechanism (O’Connor and Brian, 2000). It is not known, however, why different strains of the same TGEV or different coronaviruses evolved such distinct mechanisms in regulating the expression of the same genes.KeywordsmRNA SpeciesMouse Hepatitis VirusTransmissible Gastroenteritis VirusInternal Ribosomal Entry Site SequenceMurine CoronavirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Internal Initiation of Translation Directed by the 5′-Untranslated Region of the Tobamovirus Subgenomic RNA I2

Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRESCP,148CR). The equivalent 148-nt sequence from TMV U1 RNA (U1CP,148SP) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRESMP,228CR) contained an IRES element that directed in vitro translation of the 3′-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRESMP,228U1. It was shown that IRESMP,228CR, IRESMP,228U1, and IRESCP,148CR could mediate expression of the 3′-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRESMP,75), which corresponded closely to the length of the 5′UTR of TMV subgenomic RNA (sgRNA) I2. The RNA transcripts structurally equivalent to I2 sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRESMP,75 (HIRESMP,75CR-MP-CP-3′UTR; HIRESMP,75U1-MP-CP-3′UTR), were able to express the MP gene in vitro. The capacity of HIRESMP,75CR sequence for mediating internal translation of the 3′-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I2 sgRNAs might proceed via internal ribosome entry pathway mediated by IRESMP element contained in the 75-nt 5′UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I2 sgRNA operates concurrently.

Yellow Fever 5′ Noncoding Region as a Potential Element to Improve Hepatitis C Virus Production through Modification of Translational Control

The lengthy 5′ noncoding region (5′ NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5′ NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was comparedin vitroin rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5′ NCR. Surprisingly, the related distanced YFV 5′ NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effectivein vivoafter transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5′ NCR among theFlaviviridaefamily?

Effects of age on the posttranscriptional regulation of CCAAT/enhancer binding protein alpha and CCAAT/enhancer binding protein beta isoform synthesis in control and LPS-treated livers.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) and CCAAT/enhancer binding protein beta (C/EBPbeta) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa and C/EBPbetas of 35, 20, and approximately 8.5 kDa. The DNA-binding activities and pool levels of p42(C/EBPalpha) and p30(C/EBPalpha) in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPalpha and C/EBPbeta isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42(C/EBPalpha) levels and binding activity, whereas those of p20(C/EBPalpha) and p20(C/EBPbeta) are increased. However, translation of 42-kDa C/EBPalpha is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPalpha- and C/EBPbeta-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPalpha and C/EBPbeta isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

Alternative Translation of the Proto-oncogene c-mycby an Internal Ribosome Entry Site

The human proto-oncogene c-myc encodes two proteins, c-Myc1 and c-Myc2, from two initiation codons, CUG and AUG, respectively. It is also transcribed from four alternative promoters (P0, P1, P2, and P3), giving rise to different RNA 5'-leader sequences, the long sizes of which suggest that they must be inefficiently translated by the classical ribosome scanning mechanism. Here we have examined the influence of three c-myc mRNA 5'-leaders on the translation of chimeric myc-CAT mRNAs. We observed that in the reticulocyte rabbit lysate, these 5'-leaders lead to cap-independent translation initiation. To determine whether this kind of initiation resulted from the presence of an internal ribosome entry site (IRES), COS-7 cells were transfected with bicistronic vectors containing the different c-myc 5'-leaders in the intercistronic region. An IRES was identified, requiring elements located within the P2 leader, between nucleotides -363 and -94 upstream from the CUG start codon. This is the first demonstration of the existence of IRES-dependent translation for a proto-oncogene. This IRES could be a translation enhancer, allowing activation of c-myc expression under the control of trans-acting factors and in response to specific cell stimuli.

Characterization and Retinoic Acid Responsiveness of the Murine Hoxd4 Transcription Unit

We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group, Hoxd4. We have identified two Hoxd4 transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131-141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895-1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359-372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5'-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism.

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.

Alternative Translation of Human Fibroblast Growth Factor 2 mRNA Occurs by Internal Entry of Ribosomes

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.

CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials.

The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.