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Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5′-UTR of ASH1 mRNA and inhibited its translation in vitro. These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5′- and 3′-UTRs of ASH1 mRNA.

Translation regulation largely occurs during initiation, which features ribosome assembly onto mRNAs and selection of the translation start site. Short, upstream ORFs (uORFs) located in the 5'-leader of the mRNA can be selected for translation. Multiple transcripts associated with stress amelioration are preferentially translated through uORF-mediated mechanisms during activation of the integrated stress response (ISR) in which phosphorylation of the α subunit of eIF2 results in a coincident global reduction in translation initiation. This review presents key features of uORFs that serve to optimize translational control that is essential for regulation of cell fate in response to environmental stresses.

In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis to conserve resources and facilitate preferential translation of transcripts that promote stress adaptation. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) function to dephosphorylate eIF2α-P and restore protein synthesis. The 5'-leaders of GADD34 and CReP contain two upstream ORFs (uORFs). Using biochemical and genetic approaches we show that features of these uORFs are central for their differential expression. In the absence of stress, translation of an inhibitory uORF in GADD34 acts as a barrier that prevents reinitiation at the GADD34 coding region. Enhanced eIF2α-P during stress directs ribosome bypass of the uORF, facilitating translation of the GADD34 coding region. CReP expression occurs independent of eIF2α-P via an uORF that allows for translation reinitiation at the CReP coding region independent of stress. Importantly, alterations in the GADD34 uORF affect the status of eIF2α-P, translational control, and cell adaptation to stress. These results show that properties of uORFs that permit ribosome reinitiation are critical for directing gene-specific translational control in the integrated stress response.

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency.

Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a "scanning" mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite- inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript.

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5′ cap and a highly conserved triplet repeat sequence of the viral mRNA 5′ UTR. The simultaneous binding of N at both the terminal cap and the 5′ UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Previously, we showed that CL1-5 cells express more hypoxia-inducible factor-1alpha (HIF-1alpha) than the parental CL1 cells, which bestows CL1-5 cells a stronger invasive activity. Here, we investigated the mechanisms underlying the differential expression of HIF-1alpha mRNA in CL1 and CL1-5 cells. Data showed that the transcription rate of HIF-1alpha gene in CL1 cells was slightly higher than that of CL1-5 cells, suggesting that the expression of HIF-1alpha mRNA in CL1 cells was repressed by post-transcriptional mechanisms. RNA electrophoretic mobility shift assays revealed a 61-base segment (designated as D5) within the 5'-untranslated repeat of HIF-1alpha mRNA, with which the CL1 cell lysates formed more prominent complexes (including complex I) than did CL1-5 cell lysates. Insertion of D5 into a reporter construct reduced the half-life of the chimeric transcripts in transfected CL1 but not CL1-5 cells; conversely, overexpression of D5-containing reporter construct in CL1 cells increased HIF-1alpha mRNA. We also identified the polypyrimidine tract-binding protein (PTB) as a required component of complex I. Deletion of the RNA recognition motif 1 (RRM1) or RRM3 of PTB abolished the formation of complex I. Our data showed that CL1 cells expressed more PTB than CL1-5 cells. Inhibition of PTB expression in CL1 cells decreased the formation of complex I, whereas overexpression of PTB in CL1-5 cells increased the levels of complex I, decreased the stability of HIF-1alpha and D5-containing chimeric mRNAs, and decreased cell invasiveness. In sum, we have identified in lung adenocarcinoma cells a mechanism that regulates HIF-1alpha expression by modulating HIF-1alpha mRNA stability.

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

Cellular stress such as endoplasmic reticulum stress, hypoxia, and viral infection activates an integrated stress response, which includes the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit overall protein synthesis. Paradoxically, this leads to translation of a subset of mRNAs, like transcription factor ATF4, which in turn induces transcription of downstream stress-induced genes such as growth arrest DNA-inducible gene 34 (GADD34). GADD34 interacts with protein phosphatase 1 to dephosphorylate eIF2alpha, resulting in a negative feedback loop to recover protein synthesis and allow translation of stress-induced transcripts. Here, we show that GADD34 is not only transcriptionally induced but also translationally regulated to ensure maximal expression during eIF2alpha phosphorylation. GADD34 mRNAs are preferentially associated with polysomes during eIF2alpha phosphorylation, which is mediated by its 5'-untranslated region (5'UTR). The human GADD34 5'UTR contains two non-overlapping upstream open reading frames (uORFs), whereas the mouse version contains two overlapping and out of frame uORFs. Using 5'UTR GADD34 reporter constructs, we show that the downstream uORF mediates repression of basal translation and directs translation during eIF2alpha phosphorylation. Furthermore, we show that the upstream uORF is poorly translated and that a proportion of scanning ribosomes bypasses the upstream uORF to recognize the downstream uORF. These findings suggest that GADD34 translation is regulated by a unique 5'UTR uORF mechanism to ensure proper GADD34 expression during eIF2alpha phosphorylation. This mechanism may serve as a model for understanding how other 5'UTR uORF-containing mRNAs are regulated during cellular stress.

Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-response element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5'-untranslated regions (5'-UTRs), 5'-UTRalpha and 5'-UTRbeta, derived from exon1alpha and exon1beta. 5'-UTRalpha contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. This study was designed to investigate the potential role of 5'-UTRs in translational control. These 5'-UTRs differentially determined translation efficiency from mRNA. The presence of 5'-UTRalpha or 5'-UTRbeta represses translation from the downstream ATF5 ORF. Moreover, 5'-UTRalpha-regulated translational repression is released by amino acid limitation or NaAsO(2) exposure. This release was not seen for 5'-UTRbeta. Mutation of uAUG2 in the uORF2 of 5'-UTRalpha restored the basal expression and abolished the positive regulation by amino acid limitation or arsenite exposure. We demonstrated that phosphorylation of eukaryotic initiation factor 2alpha was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, arsenite exposure activated the exogenously expressed heme-regulated inhibitor kinase and induced the phosphorylation of eukaryotic initiation factor 2alpha in nonerythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5'-UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or arsenite-induced oxidative stress.

Little is known about the biochemical mechanism of translation in cyanobacteria though substantial studies have been made on photosynthesis, nitrogen fixation, circadian rhythm, and genome structure. To analyze the mechanism of cyanobacterial translation, we have developed an in vitro translation system from Synechococcus cells using a psbAI-lacZ fusion mRNA as a model template. This in vitro system supports accurate translation from the authentic initiation site of a variety of Synechococcus mRNAs. In Synechococcus cells, rbcL and rbcS encoding the large and small subunits, respectively, of ribulose-1,5-bisphosphate carboxylase/oxygenase are co-transcribed as a dicistronic mRNA, and the downstream rbcS mRNA possesses two possible initiation codons separated by three nucleotides. Using this in vitro system and mutated mRNAs, we demonstrated that translation starts exclusively from the upstream AUG codon. Although there are Shine-Dalgarno-like sequences in positions similar to those of the functional Shine-Dalgarno elements in Escherichia coli, mutation analysis indicated that these sequences are not required for translation. Assays with deletions within the 5'-untranslated region showed that a pyrimidine-rich sequence in the -46 to -15 region is necessary for efficient translation. Synechococcus cells contain two ribosomal protein S1 homologues of 38 and 33 kDa in size. UV cross-linking and immunoprecipitation experiments suggested that the 38-kDa S1 is involved in efficient translation via associating with the pyrimidine-rich sequence. The present in vitro translation system will be a powerful tool to analyze the basic mechanism of translation in cyanobacteria.

eIF3 in mammals is the largest translation initiation factor ( approximately 800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2alpha from complexes sedimenting at approximately 40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.

The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2alpha and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.

A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5' leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5' tiny and 3' small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5' leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.

We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered approximately 150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.

Heat shock in Drosophila results in repression of most normal (non-heat shock) mRNA translation and the preferential translation of the heat shock mRNAs. The sequence elements that confer preferential translation have been localized to the 5′-untranslated region (5′-UTR) for Hsp22 and Hsp70 mRNAs (in Drosophila). Hsp90 mRNA is unique among the heat shock mRNAs in having extensive secondary structure in its 5′-UTR and being abundantly represented in the non-heat shocked cell. In this study, we show that Hsp90 mRNA translation is inefficient at normal growth temperature, and substantially activated by heat shock. Its preferential translation is not based on an IRES-mediated translation pathway, because overexpression of eIF4E-BP inhibits its translation (and the translation of Hsp70 mRNA). The ability of Hsp90 mRNA to be preferentially translated is conferred by its 5′-UTR, but, in contrast to Hsp22 and -70, is primarily influenced by nucleotides close to the AUG initiation codon. We present a model to account for Hsp90 mRNA translation, incorporating results indicating that heat shock inhibits eIF4F activity, and that Hsp90 mRNA translation is sensitive to eIF4F inactivation.

The 5'-untranslated region (5'-UTR) of an mRNA plays an important role in translation initiation in eukaryotes. A minimal length of about 20 nucleotides is required to prevent leaky ribosome scanning. In one of the most primitive eukaryotes, Giardia lamblia, however, the mRNAs have 5'-UTRs mostly in the range of 0 to 14 nucleotides without a conserved sequence, which raises the question on how the ribosome could effectively scan such short 5'-UTRs for an accurate initiation of translation. In the present study, we expressed capped transcripts of luciferase gene in Giardia trophozoites via transfection and observed that when the 5'-UTR of the transcript was lengthened from 9 to 21 nucleotides, there was a corresponding decrease of translation efficiency. Conversely, shortening of the 5'-UTR from nine nucleotides down to a single nucleotide did not result in any reduced translation or leaky scanning. Translation appeared to initiate exclusively from the first initiation codon located downstream from the cap. Experimental evidence indicated also that a stem-loop structure immediately downstream from the initiation codon exerted significant inhibition on translation initiation when the 5'-UTR consisted of less than seven nucleotides. This inhibitory effect was abolished by increasing the distance between the stem-loop and the cap-G structure either upstream or downstream from the start codon, thus suggesting a spatial requirement for effective ribosome recruitment. Overall, our results suggest an absence of ribosome scanning for AUG in initiating translation in Giardia. A capped mRNA with a single nucleotide leader is apparently sufficient for recruiting ribosome and initiating translation.

The long and GC-rich 5'-untranslated region (5'-UTR) of the known 3.8-kb platelet-derived growth factor B (PDGF-B)/c-sis mRNA is highly conserved and inhibits its own translation. It has been thought that this 5'-UTR functions by regulating translation possibly using an internal ribosome entry site (IRES)-mediated mechanism. However, in the present study we found no evidence that the 5'-UTR sequence of PDGF-B mRNA contains any IRES activity. Instead, we found that the 5'-UTR sequence of PDGF-B functions as a promoter both constitutively and upon induction in a variety of cell lines. The 5'-UTR sequence contains two promoters (termed P1 and P2) when only the 5'-UTR sequence is analyzed. In the presence of the upstream TATA-box-containing promoter (P0), P1 and P0 promoters are integrated into one promoter, whereas the P2 promoter still functions. The full promoter with combined P0, P1, and P2 produced two transcripts, with the major one having the full-length 5'-UTR and the minor one the short 5'-UTR. The integrated P0/P1 promoter and P2 promoter are likely responsible for producing the endogenous 3.8- and 2.8-kb PDGF-B mRNAs that are detected in cultured human renal microvascular endothelial cells, a few tumor cells, and rat brain tissues. Furthermore, we detected the 2.8-kb PDGF-B mRNA in erythroleukemia K562 cells upon 12-O-tetradecanoylphorbol-13-acetate-induced differentiation. Considering that the 5'-UTR in the 3.8-kb mRNA contains no IRES activity and inhibits cap-dependent translation, we believe that the endogenous 2.8-kb mRNA produced from the 5'-UTR promoter is likely the major template responsible for protein production both constitutively and upon induction. We also found that the transcription from the 5'-UTR P2 promoter might be coordinated by the major upstream P0 promoter upon stimulation. Based on these observations, we propose that the TATA-containing P0 promoter and the 5'-UTR promoter work together to tightly control the expression of PDGF-B.

Caliciviruses represent a family of positive strand RNA viruses responsible for a variety of syndromes in man and animals. VP10, a minor structural protein of the calicivirus rabbit hemorrhagic disease virus, is encoded in the small 3'-terminal open reading frame (ORF) 2 and is translated with an efficiency of approximately 20% of the preceding ORF1. The presence of the ORF1 termination codon is crucial for VP10 expression. Translation of VP10 starts at an AUG codon located at positions -5 to -3 of the ORF1 termination codon. However, VP10 was also expressed in the absence of an AUG initiation codon. The majority of ORF1 could be deleted or replaced by different sequences without significant influence on VP10 expression as long as translation terminated at the given position. The RNA sequence of the 3'-terminal 84 nucleotides of ORF1 but not the encoded peptide was found to be crucial for VP10 expression. In contrast, nearly the entire ORF2 could be replaced by a foreign sequence without abrogation of its translation. Accordingly, VP10 is expressed in a translation termination/reinitiation process that is particular because it is independent of an AUG translational start codon and requires the presence of a sequence element upstream of the initiation site.

Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, we performed deletion and mutational analyses of an IRES contained within the 720-nucleotide (nt) 5' leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. These cis-acting sequences include a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. The 22-nt sequence was shown to function as an IRES when tested in isolation, and we demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site. The activities of all 4 cis-acting sequences were further confirmed by their mutation in the context of the full IRES. Interestingly, one of the inhibitory cis-acting sequences is contained within an upstream open reading frame (uORF), and its activity seems to be masked by translation of this uORF. Binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins. In addition, the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. The results demonstrate that different types of cis-acting sequences mediate or modulate translation of the Rbm3 mRNA and suggest that one of the IRES modules contained within the 5' leader facilitates translation initiation by binding directly to 40 S ribosomal subunits.

Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

Based on the integral role that argininosuccinate synthase (AS) plays in the production of nitric oxide in vascular endothelial cells and urea in liver, an analysis was carried out to determine whether signals reside in the AS mRNA to account for tissue differences in AS function and location. Reverse transcriptase-PCR and sequence analysis showed that the AS mRNA coding region was the same for both endothelial cells and liver; however, 5'-RACE analysis (rapid amplification of cDNA ends) identified AS mRNA species in endothelial cells in addition to a major 43-nucleotide (nt) 5'-untranslated region (UTR) AS mRNA with overlapping extended 5'-UTRs of 66 and 92 nt. Comparison to the genomic sequence immediately upstream of the reported transcription start site for the human and mouse AS gene suggested that expression of all three species of bovine endothelial AS mRNA are driven by a common promoter and that 5'-UTR diversity in endothelial cells results from three transcriptional initiation sites within exon 1. RNase protection analysis and real-time reverse transcriptase-PCR verified and quantitated the differential expression of the extended 5'-UTR species relative to the major 43-nt 5'-UTR AS mRNA. In vitro translation studies showed a less pronounced but similar discordant expression. Sequential deletions starting from the 5' terminus of the 92-nt 5'-UTR construct resulted in a corresponding increase in translational efficiency, but the most pronounced effect resulted from mutation of an upstream open reading frame, which restored translational efficiency of the 92-nt 5'-UTR AS mRNA. When the different AS mRNA 5'-UTRs, cloned in front of a luciferase reporter gene, were transfected into endothelial cells, the pattern of luciferase expression was nearly identical to that observed for the different 5'-UTR AS mRNAs in endothelial cells. Given the different roles ascribed for argininosuccinate synthase, urea versus NO production, these results suggest that sequence in the AS gene represented by position -92 to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs plays an important role in differential and tissue-specific expression.

Tumor necrosis factor-alpha (TNFalpha) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40 degrees C) markedly attenuates TNFalpha expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFalpha gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5'-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5'-UTR of the TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFalpha promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFalpha promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to an mRNA. Plants, animals, and yeast each express two eIF4G homologs, which share only 30, 46, and 53% identity, respectively. We have examined the functional differences between plant eIF4G proteins, referred to as eIF4G and eIFiso4G, when present as subunits of eIF4F and eIFiso4F, respectively. The degree to which a 5'-cap stimulated translation was inversely correlated with the concentration of eIF4F or eIFiso4F and required the poly(A)-binding protein for optimal function. Although eIF4F and eIFiso4F directed translation of unstructured mRNAs, eIF4F supported translation of an mRNA containing 5'-proximal secondary structure substantially better than did eIFiso4F. Moreover, eIF4F stimulated translation from uncapped monocistronic or dicistronic mRNAs to a greater extent than did eIFiso4F. These data suggest that at least some functions of plant eIFiso4F and eIF4F have diverged in that eIFiso4F promotes translation preferentially from unstructured mRNAs, whereas eIF4F can promote translation also from mRNAs that contain a structured 5'-leader and that are uncapped or contain multiple cistrons. This ability may also enable eIF4F to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited.

Fragile X syndrome, the leading heritable form of mental impairment, is generally caused by large expansions of a CGG repeat in the promoter region of the FMR1 gene followed by transcriptional silencing. However, there is growing evidence that translation of the FMR1 message is also impaired, presumably because of the expanded CGG element in the 5'-untranslated region (5'-UTR) of the FMR1 message. To study the properties of the FMR1 5'-UTR, deletions were generated within a normal 5'-UTR with 16 CGG repeats for both monocistronic and dicistronic (luciferase) reporter constructs. Transient transfection experiments revealed a approximately 20-nucleotide region upstream of the CGG repeat element that functions as an internal ribosome entry site (IRES). The normal CGG element itself does not appear to influence the efficiency of IRES-mediated stimulation of downstream reporter activity (approximately 18-fold over controls). Additional controls indicate that the enhanced activity of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The role of the FMR1 IRES element is not known at present; however, by analogy to other IRES-containing mRNAs expressed in neurons, the FMR1 IRES element may help to promote translation in dendrites.

The insulin-like growth factor I receptor (IGF-IR) is a heterotetrameric receptor mediating the effects of insulin-like growth I and other growth factors. This receptor is encoded by an mRNA containing an unusually long, G-C-rich, and highly structured 5' untranslated region. Using bicistronic constructs, we demonstrated here that the 5' untranslated region of the IGF-IR allows translation initiation by internal ribosome entry and therefore constitutes an internal ribosome entry site. In vitro cross-linking revealed that this internal ribosome entry site binds a protein of 57 kDa. Immunoprecipitation of UV cross-linked proteins proved that this protein was the polypyrimidine tract-binding protein, a well known regulator of picornavirus mRNA translation. The efficiency of translation of the endogenous IGF-IR mRNA is not affected by rapamycin, which is a potent inhibitor of cap-dependent translation. This result provides evidence that the endogenous IGF-IR mRNA is translated, at least in part, through a cap-independent mechanism. This is the first report of a growth factor receptor containing sequence elements that allow translation initiation to occur by internal initiation. Because the IGF-IR has a pivotal function in the cell cycle, this mechanism of translation regulation could play a crucial role in the control of cell proliferation and differentiation.

Cell surface receptor activation generally leads to changes in mRNA abundance, which may involve regulatory targets in processes working at the post-transcriptional level. Many types of agonists down-regulate vascular smooth muscle angiotensin receptor (AT(1)-R) gene expression, but it is unclear which of these activate post-transcriptional mechanisms. To reconstitute faithfully the normal AT(1)-R mRNA regulatory environment, tetracycline-suppressible promoters drive highly accurate recombinant AT(1)-R mRNA mimics in vascular smooth muscle cells that co-express an endogenous AT(1)-R mRNA. Down-regulation of the latter occurs shortly after stimulating mitogenic receptors or by using forskolin, but only cAMP signaling reduces expression of the recombinant AT(1)-R mRNA. Transcription of the recombinant mRNA is unaffected by cAMP signaling. Deletions of the AT(1)-R mRNA 3'-untranslated region do not impair cAMP-mediated down-regulation. Both loss of function and gain of function mutants show the response is mediated by the 5'-untranslated region. These observations provide the first direct functional evidence for modulation of vascular AT(1)-R gene expression by a mechanism involving a protein kinase A-regulated post-transcriptional process.

Overexpression of the HER-2 (neu, erb B-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the beta-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1alpha and IL-1beta increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated region (5'-UTR) conferred translational regulation by IL-1alpha and IL-1beta to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is homologous to related translational control elements in the 5'-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.

The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5'-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that three cis-regulatory elements located within the region from -89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5'-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2.

The upstream open reading frame (uORF) in the 5' leader of the mammalian mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) serves as a negative regulatory element by suppressing translation of the associated downstream cistron. Certain changes in the amino acid sequence of the hexapeptide (sequence MAGDIS) encoded by the uORF destroy suppressive activity, implying specific interaction with a cellular target. In this paper, we examine the extent of alterations that can be tolerated in this uORF. The mammalian AdoMetDC uORF inhibits downstream translation when placed into the 5' leader of a yeast mRNA with characteristics resembling those in mammalian cells, suggesting that the encoded peptide has a similar target across species. Using yeast for the initial screen, we tested the specificity of the critical three codons at the 3' end of the uORF by saturation mutagenesis. Altered uORFs selected from the primary yeast screen were then retested in mammalian cells. The requirements at codons 4 and 5 were quite stringent; only aspartic acid at codon 4 yielded a fully suppressive peptide, and only valine could substitute productively for isoleucine at codon 5. The specificity at codon 6 was much looser, with many substitutions retaining suppressive activity in both yeast and mammalian cells.

AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

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.

Cap proximity is a requirement to enable secondary structures and RNA-binding proteins to repress translational initiation via the 5'-untranslated region (5'-UTR) of mammalian mRNAs. We show that in Saccharomyces cerevisiae, unlike mammalian cells, the in vitro translational repressive effect of the mammalian iron regulatory protein 1 (IRP1) is independent of the site of its target in the 5'-UTR, the iron-responsive element (IRE). In vitro studies demonstrate that the binding affinity of IRP1 is also unaffected by the position of the IRE. Using IRE loop mutants, we observe an almost complete loss of IRP1-dependent repression in yeast concomitant with a 150-fold reduction in binding affinity for the IRE target. This mirrors the natural quantitative range of iron-induced adjustment of IRE/IRP1 affinity in mammalian cells. By enhancing the stability of the IRE stem-loop, we also show that its intrinsic folding energy acts together with the binding energy of IRP1 to give an additive capacity to restrict translational initiation. An IRE.IRP1 complex in a cap-distal position in yeast blocks scanning 40 S ribosomes on the 5'-UTR. It follows that the position effect of mammalian site-specific translational repression is dictated by the competence of the mammalian preinitiation complex to destabilize inhibitory structures at different steps of the initiation process.

Pituitary adenylate cyclase-activating polypeptide (PACAP) elicits its diverse biological actions by interacting with both PACAP-selective type I PACAP receptors (PACAPRs) and type II PACAPRs that do not distinguish between PACAP and vasoactive intestinal polypeptide. Using long distance polymerase chain reaction, we amplified and characterized the entire coding region of the rat type I PACAPR (rPACAPR) gene, which spans 40 kilobases and contains 15 exons. Mapping of the exons and sequencing of all intron-exon boundaries revealed a structural organization of the rPACAPR gene that is very similar to those encoding other members of the calcitonin/secretin/parathyroid hormone receptor family. Southern blot analysis demonstrated a single copy of the rPACAPR gene. A combination of rapid amplification of cDNA ends and reverse transcriptase polymerase chain reaction revealed an unexpected diversity in the rPACAPR mRNA in the 5'-untranslated (5'-UTR) region. Four rPACAPR cDNAs were identified with 5'-UTR sequences that all diverged from the genomic sequence at a site 76 bp upstream of the ATG start codon, where a consensus 3' slice acceptor sequence was located. Sequence analysis of these amplified transcripts demonstrated that they arise by tissue-specific differential usage of four exons in the 5' noncoding region of the rPACAPR gene. This study is the first to elucidate the structural organization of a PACAPR gene and to demonstrate that alternative splicing generates rPACAPR transcripts with unique 5'-UTRs.

Folate receptors (FR) are inversely regulated by the extracellular folate concentration at the translational level in cervical carcinoma cells. Accordingly, the potential for interaction of cis-elements in FR-α mRNA and trans-factors in these cells was determined. Gel-shift assays identified two signals that were specifically derived from the interaction of cytosolic proteins with the 5′-untranslated region of FR-α mRNA. RNase T1 mapping revealed that the RNA sequences interacting with these proteins were located between nucleotides −133 to −116 (18-bases) and −158 to −116 (43-bases), upstream of the translation start site. However, selective RNase H cleavage indicated that the 18-base RNA sequence was the cis-element. The RNA-protein interaction was competed by poly(C), but not by poly(U), homopolymers. UV cross-linking and Northwestern blot analysis confirmed that the trans-factors were 46-kDa proteins. An 18-base antisense oligodeoxynucleotide complementary to the cis-element specifically quenched the RNA-protein interaction and also completely inhibited translation of FR-α mRNA without changing its stability. Thus, the interaction of the 18-base cis-element and the 46-kDa trans-factors likely have an important role in translational regulation of FR. In addition, because the 46-kDa proteins were widely distributed in cells expressing little to no FR-α, these species probably have additional functions that are unrelated to translation of FR.

A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5'-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5'-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5'-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation.

A small upstream open reading frame (uORF), located 14 nucleotides from the cap in the 5' transcript leader (5' TL) of the mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC), suppresses translation of the downstream cistron in normal T cells and T cell lines. In the present study, we examined the structural features of the 5' TL that overcome this suppressive influence in cells of nonlymphoid origin. Initiation at the downstream cistron in nonlymphoid cells is by a cap-dependent mechanism that requires ribosome scanning along the 5' TL and does not involve an internal ribosome entry site. Extending the uORF so that it overlapped the major cistron by 101 nucleotides had no effect on translation of the downstream cistron in either HeLa or Jurkat cells. When the distance between the uORF and the cap was extended to 47 nucleotides, using sequence previously found to be neutral, translation of the major cistron was inhibited 5-fold in HeLa cells and the mRNA was moved from polysomes to monosomes, a location identical to that of the wild type mRNA in Jurkat cells. Therefore, in contrast to T cells, initiation at the uORF seems to be relatively infrequent in non-lymphoid cells due to its proximity to the cap, allowing efficient translation of the downstream cistron.

The influence of 5'-secondary structure formation and 5'-leader length upon mRNA translation in yeast has been analyzed using a closely related set of cat mRNAs (Vega Laso, M. R., Zhu, D., Sagliocco, F. A., Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462). A cat mRNA with a relatively short unstructured 5'-leader (22 bases) had a ribosome loading about half that of a cat mRNA with an unstructured 5'-leader of 77 bases. The introduction of 5'-secondary structures at various positions throughout the 5'-leader of the cat mRNA inhibited translation initiation, the degree of inhibition being largely dependent upon the thermodynamic stability of the structure. Each mRNA carrying a 5'-secondary structure had a biphasic polysome distribution, indicating that the mRNA molecules were distributed between untranslated and well translated subpopulations. This suggests that once 5'-secondary structures are unwound, they reform slowly relative to the rate of translation initiation in yeast. Untranslated mRNA accumulated in 43 S preinitiation complexes, even when there were only 5 bases between the 5'-cap and the base of the hairpin. The data are consistent with the scanning hypothesis (Kozak, M. (1989) J. Cell. Biol. 108, 229-241) and suggest that 40 S ribosomal subunits bind to mRNA early in the scanning process, probably before mRNA unwinding has taken place.

We found that a rat testis cDNA library contained two types of proenkephalin cDNA, corresponding to somatic and germ cell-specific transcripts. The somatic form is identical in primary sequence to previously characterized transcripts from rat brain and heart. The longer germ cell-specific cDNA contains a novel 5' terminus derived from the somatic intron A. We found that germ cell-specific transcripts translate in vitro with significantly lower efficiency than do somatic-type transcripts. Deletion analysis indicated that translational inefficiency is mediated primarily by the presence of four upstream AUG codons followed by short open reading frames within the leader sequence. Secondary structure in the form of a stem-and-loop upstream of the initiator codon for proenkephalin was found to have a slightly inhibitory effect on translation. These results, coupled with the very low abundance of the somatic transcript, provide a possible explanation for the low levels of opioid peptides found in adult rat testis in spite of the abundant quantity of germ cell-specific proenkephalin transcript.

In cells infected by influenza virus type A, host cell protein synthesis declines rapidly and dramatically, while influenza viral protein synthesis occurs efficiently throughout infection. Previously, we had shown that the selective translation of influenza viral mRNAs in infected cells occurred in a cap-dependent manner and was due at least in part to structures inherent in the mRNAs. Using chimeras containing the noncoding and coding regions of cellular and viral mRNAs, we can now report that the selective translation is mediated by sequences within the 5'-untranslated regions (UTR) of the viral mRNAs. Polysome analysis confirmed that a 45-nucleotide sequence contained in the 5'-UTR of the influenza viral nucleocapsid protein was necessary and sufficient to allow the host cell translational machinery to discriminate between viral and cellular mRNAs. In reciprocal experiments in which the 5'-UTR of the cellular mRNA-secreted embryonic alkaline phosphatase replaced the nucleocapsid protein 5'-UTR, viral protein synthesis was inhibited in virus-infected cells, resembling host protein synthesis. Finally, we demonstrated that the 5'-UTR of another influenza viral mRNA, that encoding the nonstructural protein, also conferred resistance to the shutoff of protein synthesis in influenza virus-infected cells.

The mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) has a 330-nucleotide 5'-transcript leader containing an open reading frame (uORF) that codes for the hexapeptide MAGDIS. The uORF restricts the intracellular distribution of AdoMetDC mRNA primarily to monosomes in normal T-lymphocytes and in T-cell lines. In contrast, non-lymphoid cells normally carry an average of seven to nine ribosomes per AdoMetDC mRNA molecule (Hill, J.R., and Morris, D. R. (1992) J. Biol. Chem. 267, 21886-21893). Several alterations abolish the negative regulatory effect of the uORF in T-cells. These include removing the site of translational initiation; weakening the context of the translational initiation site; changing the coding capacity of the fourth, fifth, and sixth codons; increasing the length of the uORF at either the 5' or 3' end; or changing the primary order of the codons. In contrast, altering the nucleic acid sequence of the uORF at degenerative positions without changing the amino acid coding capacity did not cause deregulation. The uORF does not regulate translation in the trans-configuration. Our results support a model in which translation of the uORF generates a nascent hexapeptide that interacts with its translating ribosome to suppress translation of AdoMetDC mRNA in a cell-specific manner. Structural features of the carboxyl-terminal 3 amino acids of the putative hexapeptide govern the interaction of the peptide with a component of the translation machinery.

Expression of the Na,K-ATPase alpha and beta subunit genes is influenced by a complex series of regulatory pathways. For example, unequal amounts of subunit mRNAs are detected in several tissues, both at rest and upon mRNA induction, even though equal quantities of subunit proteins exist. We therefore studied mRNA stability and translational efficiency of wild type, deletion mutant, and chimeric subunit mRNAs in a cell-free translation system, to examine the possible role of post-transcriptional events in regulating subunit expression. alpha 1 mRNA translated less efficiently than beta 1 mRNA and competed less efficiently for rate-limiting translation factors. Deletion of the 5'-untranslated region of alpha 1 mRNA significantly increased its translation efficiency. Conversely, the alpha 1 5'-untranslated region impaired translation of beta 1 mRNA when attached upstream of the beta 1 coding sequence. This region is G/C-rich, and has a complex mRNA secondary structure. We propose that differential translational efficiency may contribute to the equal biosynthesis of subunit proteins in those tissues in which subunit mRNAs either exist in unequal amounts or are differentially induced.

An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.

The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA.

Although steroids have been implicated in post-transcriptional regulation, their effects on mRNA translation rates have been uncertain. We have used a cell-free translation system programmed with synthetic messages to show that steroids can alter the translation rates of a number of myelin protein mRNAs and the mRNA encoding a non-myelin protein, the estrogen receptor. Through the use of deletion analysis, site-directed mutagenesis, and chimeric mRNAs we have identified a 9-nucleotide segment in the 5'-untranslated region of one myelin protein mRNA that is necessary for steroid action. Steroid-mediated translational regulation is discussed in terms of myelination where subtle developmental changes in protein composition of the membrane have significant consequences on its morphology and function. We propose that the modulation of mRNA translation rates by steroids is a more general phenomenon that may serve as another mechanism by which steroids can regulate gene expression.

During the differentiation of C2 myoblasts into differentiated myotubes, there is a marked decrease in the abundance of c-myc mRNA. c-myc transcription initiation and elongation do not change significantly, but the turnover of c-myc transcripts appears to be accelerated during differentiation. We examined the expression of several recombinant c-myc genes introduced into C2 cells by stable transfection and found that mRNAs containing the c-myc protein-coding region of exons 2 and 3 are appropriately regulated; the upstream c-myc sequences, the long leader sequence encoded by c-myc exon 1, and the 3' untranslated region are dispensible for proper regulation. Regulation appears to affect c-myc transcripts that are bound to polyribosomes, and the mRNA from a gene with a point mutation in the translation initiation codon is no longer properly regulated. We conclude that down-regulation of c-myc expression during myogenesis is post-transcriptional and requires the presence of sequences encoding the c-myc protein in a form that can be translated.