Translation acrobatics: how cancer cells exploit alternate modes of translational initiation

Recent work has brought to light many different mechanisms of translation initiation that function in cells in parallel to canonical cap‐dependent initiation. This has important implications for cancer. Canonical cap‐dependent translation initiation is inhibited by many stresses such as hypoxia, nutrient limitation, proteotoxic stress, or genotoxic stress. Since cancer cells are often exposed to these stresses, they rely on alternate modes of translation initiation for protein synthesis and cell growth. Cancer mutations are now being identified in components of the translation machinery and in cis‐regulatory elements of mRNAs, which both control translation of cancer‐relevant genes. In this review, we provide an overview on the various modes of non‐canonical translation initiation, such as leaky scanning, translation re‐initiation, ribosome shunting, IRES‐dependent translation, and m⁶A‐dependent translation, and then discuss the influence of stress on these different modes of translation. Finally, we present examples of how these modes of translation are dysregulated in cancer cells, allowing them to grow, to proliferate, and to survive, thereby highlighting the importance of translational control in cancer.     

eIF5A binds to translational machinery components and affects translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archebacteria to mammals. Although this protein was originally identified as a translation initiation factor, subsequent experiments did not support a role for eIF5A in general translation. In this work, we demonstrate that eIF-5A interacts with structural components of the 80S ribosome, as well as with the translation elongation factor 2 (eEF2). Moreover, eIF5A is further shown to cofractionate with monosomes in a translation-dependent manner. Finally, eIF5A mutants show altered polysome profiles and are sensitive to translation inhibitors. Our results re-establish a function for eIF5A in translation and suggest a role for this factor in translation elongation instead of translation initiation.     

The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.

Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.     

The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex

The translation, localization, and degradation of cytoplasmic mRNAs are controlled by the formation and rearrangement of their mRNPs. The conserved Ded1/DDX3 DEAD-box protein functions in an unknown manner to affect both translation initiation and repression. We demonstrate that Ded1 first functions by directly interacting with eIF4G to assemble a Ded1-mRNA-eIF4F complex, which accumulates in stress granules. After ATP hydrolysis by Ded1, the mRNP exits stress granules and completes translation initiation. Thus, Ded1 functions both as a repressor of translation, by assembling an mRNP stalled in translation initiation, and as an ATP-dependent activator of translation, by resolving the stalled mRNP. These results identify Ded1 as a translation initiation factor that assembles and remodels an intermediate complex in translation initiation.     

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.     

Facile characterization of translation initiation via nonsense codon suppression.

A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.     

Translational regulation of Hsp90 mRNA. AUG-proximal 5'-untranslated region elements essential for preferential heat shock translation

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.     

Analysis and prediction of translation rate based on sequence and functional features of the mRNA

Protein concentrations depend not only on the mRNA level, but also on the translation rate and the degradation rate. Prediction of mRNA's translation rate would provide valuable information for in-depth understanding of the translation mechanism and dynamic proteome. In this study, we developed a new computational model to predict the translation rate, featured by (1) integrating various sequence-derived and functional features, (2) applying the maximum relevance & minimum redundancy method and incremental feature selection to select features to optimize the prediction model, and (3) being able to predict the translation rate of RNA into high or low translation rate category. The prediction accuracies under rich and starvation condition were 68.8% and 70.0%, respectively, evaluated by jackknife cross-validation. It was found that the following features were correlated with translation rate: codon usage frequency, some gene ontology enrichment scores, number of RNA binding proteins known to bind its mRNA product, coding sequence length, protein abundance and 5'UTR free energy. These findings might provide useful information for understanding the mechanisms of translation and dynamic proteome. Our translation rate prediction model might become a high throughput tool for annotating the translation rate of mRNAs in large-scale.     

Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides

The translation of numerous eukaryotic mRNAs is mediated by internal ribosomal entry sites (IRESs). IRES-dependent translation requires both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs). Here we report a strategy to identify and characterize ITAFs required for IRES-dependent translation. This process involves steps for identifying oligodeoxynucleotides affecting IRES-dependent translation, purifying proteins interacting with the inhibitory DNA, identifying the specific proteins with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and confirming the roles of these proteins in IRES-dependent translation by depletion and repletion of proteins from an in vitro translation system. Using this strategy, we show that poly(rC)-binding proteins 1 and 2 enhance translation through polioviral and rhinoviral IRES elements.     

Target-specific arrest of mRNA translation by antisense 2'-O-alkyloligoribonucleotides.

We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNA-hybrids positioned anywhere within the 5' UTR or the first approximately 20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-O-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralen-tagged 2'-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-O-alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.     

A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation

A significant number of viral and cellular mRNAs utilize cap-independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap-independent translation requires noncanonical, cellular RNA-binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo-cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)-mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA-binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES-mediated translation), further documenting the multifunctional nature of this important class of cellular RNA-binding proteins.     

DAP5 associates with eIF2β and eIF4AI to promote Internal Ribosome Entry Site driven translation

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5′UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2β and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.     

Pioneer round of translation occurs during serum starvation

The pioneer round of translation plays a role in translation initiation of newly spliced and exon junction complex (EJC)-bound mRNAs. Nuclear cap-binding protein complex CBP80/20 binds to those mRNAs at the 5'-end, recruiting translation initiation complex. As a consequence of the pioneer round of translation, the bound EJCs are dissociated from mRNAs and CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E. Steady-state translation directed by eIF4E allows for an immediate and rapid response to changes in physiological conditions. Here, we show that nonsense-mediated mRNA decay (NMD), which restricts only to the pioneer round of translation but not to steady-state translation, efficiently occurs even during serum starvation, in which steady-state translation is drastically abolished. Accordingly, CBP80 remains in the nucleus and processing bodies are unaffected in their abundance and number in serum-starved conditions. These results suggest that mRNAs enter the pioneer round of translation during serum starvation and are targeted for NMD if they contain premature termination codons.