Despite a recent surge of reports about how microRNAs might regulate translation, the question has not been answered. The proposed mechanisms contradict one another, and none is supported by strong evidence. This review explains some deficiencies in the experiments with microRNAs. Some of the problems are traceable to bad habits carried over from older studies of translational regulation, here illustrated by discussing two models involving mRNA binding proteins. One widely-accepted model, called into doubt by recent findings, is the maskin hypothesis for translational repression of cyclin B1 in Xenopus oocytes. The second dubious model postulates repression of translation of ceruloplasmin by mRNA binding proteins. A big fault in the latter case is reconstructing the imagined mechanism before looking carefully at the real thing-a criticism that applies also to studies with microRNAs. Experiments with microRNAs often employ internal ribosome entry sequences (IRESs) as tools, necessitating brief discussion of that topic. A sensitive new assay reveals that many putative IRESs promote expression of downstream cistrons via splicing rather than internal initiation of translation. Recent claims about the biological importance of IRES-binding proteins-including suggestions that these proteins might serve as targets for cancer therapy-are not supported by any meaningful evidence. The bottom line is that older studies of mRNA binding proteins and putative IRESs have created a confusing picture of translational regulation which is not helpful when trying to understand how microRNAs might work. The obvious biological importance of microRNAs makes it essential to understand how they do what they do. Fresh ways of thinking and looking are needed.

Some popular ideas about translational regulation in eukaryotes have been recognized recently as mistakes. One example is the rejection of a long-standing idea about involvement of S6 kinase in translation of ribosomal proteins. Unfortunately, new proposals about how S6 kinase might regulate translation are based on evidence that is no better than the old. Recent findings have also forced rejection of some popular ideas about the function of sequences at the 3' end of viral mRNAs and rejection of some ideas about internal ribosome entry sequences (IRESs). One long-held belief was that tissue-specific translation via an IRES underlies the neurotropism of poliovirus and the attenuation of Sabin vaccine strains. Older experiments that appeared to support this belief and recent experiments that refute it are discussed. The hypothesis that dyskeratosis congenita is caused by a defect in IRES-mediated translation is probably another mistaken idea. The supporting evidence, such as it is, comes from a mouse model of the disease and is contradicted by studies carried out with cells from affected patients. The growing use of IRESs as tools to study other questions about translation is discussed and lamented. The inefficient function of IRESs (if they are IRESs) promotes misunderstandings. I explain again why it is not valid to invoke a special mechanism of initiation based on the finding that edeine (at very low concentrations) does not inhibit the translation of a putative IRES from cricket paralysis virus. I explain why new assays, devised to rule out splicing in tests with dicistronic vectors, are not valid and why experiments with IRESs are not a good way to investigate the mechanism whereby microRNAs inhibit translation.

Selection of the translational initiation site in most eukaryotic mRNAs appears to occur via a scanning mechanism which predicts that proximity to the 5' end plays a dominant role in identifying the start codon. This "position effect" is seen in cases where a mutation creates an AUG codon upstream from the normal start site and translation shifts to the upstream site. The position effect is evident also in cases where a silent internal AUG codon is activated upon being relocated closer to the 5' end. Two mechanisms for escaping the first-AUG rule--reinitiation and context-dependent leaky scanning--enable downstream AUG codons to be accessed in some mRNAs. Although these mechanisms are not new, many new examples of their use have emerged. Via these escape pathways, the scanning mechanism operates even in extreme cases, such as a plant virus mRNA in which translation initiates from three start sites over a distance of 900 nt. This depends on careful structural arrangements, however, which are rarely present in cellular mRNAs. Understanding the rules for initiation of translation enables understanding of human diseases in which the expression of a critical gene is reduced by mutations that add upstream AUG codons or change the context around the AUG(START) codon. The opposite problem occurs in the case of hereditary thrombocythemia: translational efficiency is increased by mutations that remove or restructure a small upstream open reading frame in thrombopoietin mRNA, and the resulting overproduction of the cytokine causes the disease. This and other examples support the idea that 5' leader sequences are sometimes structured deliberately in a way that constrains scanning in order to prevent harmful overproduction of potent regulatory proteins. The accumulated evidence reveals how the scanning mechanism dictates the pattern of transcription--forcing production of monocistronic mRNAs--and the pattern of translation of eukaryotic cellular and viral genes.

Some diseases are caused by mutations that perturb the initiation step of translation by changing the context around the AUG(START) codon or introducing upstream AUG codons. The scanning mechanism provides a framework for understanding the effects of these and other structural changes in mRNAs derived from oncogenes, tumor suppressor genes, and other key regulatory genes. In mRNAs from mutated as well as normal genes, translation sometimes initiates from an internal AUG codon. Sanctioned mechanisms that allow this, including leaky scanning and reinitiation, are discussed. Thrombopoietin mRNA is an example in which translation normally initiates from an internal position via an inefficient reinitiation mechanism. Mutations that restructure this mRNA in ways that elevate production of thrombopoietin cause hereditary thrombocythemia, demonstrating that some mRNAs are designed deliberately with upstream AUG codons to preclude efficient translation and thus to prevent harmful overproduction of potent proteins. While upstream AUG codons in certain mRNAs thus play an important regulatory role, the frequency of upstream AUG codons tends to be exaggerated when cDNA sequences are compiled and analyzed. Because the discovery of mutations that perturb translation usually begins with cDNA analysis, some misunderstandings vis-a-vis the interpretation of cDNA sequences are discussed.

The belief that initiation of translation requires communication between the 5' and 3' ends of the mRNA guides--or misguides--the interpretation of many experiments. The closed-loop model for initiation creates the expectation that sequences at the 3' end of eukaryotic mRNAs should regulate translation. This review looks closely at the evidence in three prominent cases where such regulation is claimed. The mRNAs in question encode 15-lipoxygenase, ceruloplasmin, and histones. Vertebrate histone mRNAs lack a poly(A) tail, instead of which a 3' stem-loop structure is said to promote translation by binding a protein which purportedly binds initiation factors. The proffered evidence for this hypothesis has many flaws. Temporal control of 15-lipoxygenase production in reticulocytes is often cited as another well-documented example of translational regulation via the 3' untranslated region, but inspection of the evidence reveals significant gaps and contradictions. Solid evidence is lacking also for the idea that a ribosomal protein binds to and shuts off translation of ceruloplasmin mRNA. Some viral RNAs that lack a poly(A) tail have alternative 3' structures which are said to promote translation via circularization of the mRNA, but in no case has this been shown convincingly. Interpretation of many experiments is compromised by possible effects of the 3' structures on mRNA stability rather than translation. The functional-half-life assay, which is often employed to rule out effects on mRNA stability, might not be adequate to settle the question. Other issues, such as the possibility of artifacts caused by overexpression of RNA-binding proteins, can complicate studies of translational regulation. There is no doubt that elements at the 3' end of eukaryotic mRNAs can regulate gene expression in a variety of ways. It has not been shown unequivocally that one of these ways involves direct participation of the 3' untranslated region in the initiation step of translation.

Real progress in understanding translational regulatory mechanisms lags behind the claims of progress. Novel mechanisms were proclaimed in recent months for some important regulatory proteins from Drosophila (e.g. Bruno, Sex-lethal, Reaper), but the evidence is thin. Many flaws in the design and interpretation of new experiments can be traced to older experiments which came to be accepted, not because the evidence was overwhelming, but because the ideas were appealing. Two of these classic examples of translational regulation are discussed before taking up the newer findings. One paradigm concerns regulation of 15-lipoxygenase production during reticulocyte maturation. The mechanism postulated for 15-lipoxygenase was pieced together in vitro and has never been linked in a meaningful way to what happens naturally in reticulocytes; nevertheless, these experiments have guided (or misguided) thinking about how sequences near the 3' end of an mRNA might regulate translation. The second paradigm concerns the regulation of cyclin B1 translation in Xenopus oocytes by a protein called Maskin, which purportedly interacts with initiation factors. A third topic discussed in some detail concerns the idea that in eukaryotes, as in prokaryotes, initiation of translation might involve base-pairing between mRNA and ribosomal RNA. Recent experiments undertaken to test this idea in yeast are far from conclusive. Many of the experimental defects brought to light in this review are simple-absence of controls, reliance on indirect tests, failure to test a new test system before using it; these things are fixable. Special problems are posed by the practice of using internal ribosome entry sequences (IRESs) as tools to figure out how translation might be regulated by other components. Unanswered questions about the IRESs themselves have to be resolved before they can be used confidently as tools.

This review takes a second look at a set of mRNAs that purportedly employ an alternative mechanism of initiation when cap-dependent translation is reduced during mitosis or stress conditions. A closer look is necessary because evidence cited in support of the internal initiation hypothesis is often flawed. When putative internal ribosome entry sequences (IRESs) are examined more carefully, they often turn out to harbor cryptic promoters or splice sites. This undermines the dicistronic assay, wherein IRES activity is measured by the ability to support translation of the 3' cistron. Most putative IRESs still have not been checked carefully to determine whether the dicistronic vector produces only the intended dicistronic mRNA. The widespread use of the pRF vector is a major problem because this vector, which has Renilla luciferase as the 5' cistron and firefly luciferase as the 3' cistron, has been found to generate spliced transcripts. RNA transfection assays could theoretically circumvent these problems, but most candidate IRESs score very weakly in that test. The practice of calling even very weak results 'positive' is one of the problems discussed herein. The extremely low efficiency of putative IRESs is inconsistent with their postulated biological roles.'...if it is a Miracle, any sort of evidence will answer, but if it is a Fact, proof is necessary'-Mark Twain, Letters from the Earth.

Despite a recent surge of reports about how microRNAs might regulate translation, the question has not been answered. The proposed mechanisms contradict one another, and none is supported by strong evidence. This review explains some deficiencies in the experiments with microRNAs. Some of the problems are traceable to bad habits carried over from older studies of translational regulation, here illustrated by discussing two models involving mRNA binding proteins. One widely-accepted model, called into doubt by recent findings, is the maskin hypothesis for translational repression of cyclin B1 in Xenopus oocytes. The second dubious model postulates repression of translation of ceruloplasmin by mRNA binding proteins. A big fault in the latter case is reconstructing the imagined mechanism before looking carefully at the real thing-a criticism that applies also to studies with microRNAs. Experiments with microRNAs often employ internal ribosome entry sequences (IRESs) as tools, necessitating brief discussion of that topic. A sensitive new assay reveals that many putative IRESs promote expression of downstream cistrons via splicing rather than internal initiation of translation. Recent claims about the biological importance of IRES-binding proteins-including suggestions that these proteins might serve as targets for cancer therapy-are not supported by any meaningful evidence. The bottom line is that older studies of mRNA binding proteins and putative IRESs have created a confusing picture of translational regulation which is not helpful when trying to understand how microRNAs might work. The obvious biological importance of microRNAs makes it essential to understand how they do what they do. Fresh ways of thinking and looking are needed.

Translation of some mRNAs is postulated to occur via an internal initiation mechanism which is said to be augmented by a variety of RNA-binding proteins. A pervasive problem is that the RNA sequences to which the proteins bind were not rigorously proven to function as internal ribosome entry sites (IRESs). Critical examination of the evidence reveals flaws that leave room for alternative interpretations, such as the possibility that IRES elements might function as cryptic promoters, splice sites, or sequences that modulate cleavage by RNases. The growing emphasis on IRES-binding proteins diverts attention from these fundamental unresolved issues. Many of the putative IRES-binding proteins are heterogeneous nuclear ribonucleoproteins that have recognized roles in RNA processing or stability and no recognized role in translation. Thus the mechanism whereby they promote internal initiation, if indeed they do, is not obvious. Some recent experiments were said to support the idea that IRES-binding proteins cause functionally important changes in folding of the RNA, but the evidence is not convincing when examined closely. The proteins that bind to some (not all) viral IRES elements include a subset of authentic initiation factors. This has not been demonstrated with any candidate IRES of cellular origin, however; and even with viral RNAs, the required chase experiment has not been done to prove that a pre-bound initiation factor actually mediates subsequent entry of ribosomes. In short, the focus on IRES-binding proteins has gotten us no closer to understanding the mechanism of internal initiation. Given the aforementioned uncertainty about whether other mechanisms (splicing, cryptic promoters) might underlie what-appears-to-be internal initiation, a temporary solution might be to redefine IRES to mean "internal regulatory expression sequence." This compromise would allow the sequences to be used for gene expression studies, for which they sometimes work, without asserting more than has been proven about the mechanism.

This review discusses the need to re-examine some popular but unproven ideas about regulation of translation in eukaryotes. Translational control is invoked often on superficial grounds, such as a discrepancy between mRNA and protein levels which could be explained instead by rapid turnover of the protein. It is essential to verify that there is translational control (i.e., essential to rule out alternative mechanisms) before asking how translation is regulated. Many of the postulated control mechanisms are dubious. It is easy to create artifactual regulation (a slight increase or decrease in translation) by over-expressing recombinant RNA-binding proteins. The internal-initiation hypothesis is the source of other misunderstandings. Recent claims about the involvement of internal ribosome entry sequences (IRESs) in cancer and other diseases are discussed. The scanning model for initiation provides a more credible framework for understanding many aspects of translation, including ways to restrict the production of potent regulatory proteins which would be harmful if over-expressed. The rare production in eukaryotes of dicistronic mRNAs (e.g., from retrotransposons) raises questions about how the 3' cistron gets translated. Some proposed mechanisms are discussed, but the available evidence does not allow resolution of the issue. J. Cell. Biochem.(c) 2007 Wiley-Liss, Inc.

The mechanism of initiation of translation differs between prokaryotes and eukaryotes, and the strategies used for regulation differ accordingly. Translation in prokaryotes is usually regulated by blocking access to the initiation site. This is accomplished via base-paired structures (within the mRNA itself, or between the mRNA and a small trans-acting RNA) or via mRNA-binding proteins. Classic examples of each mechanism are described. The polycistronic structure of mRNAs is an important aspect of translational control in prokaryotes, but polycistronic mRNAs are not usable (and usually not produced) in eukaryotes. Four structural elements in eukaryotic mRNAs are important for regulating translation:(i) the m7G cap;(ii) sequences flanking the AUG start codon;(iii) the position of the AUG codon relative to the 5' end of the mRNA; and (iv) secondary structure within the mRNA leader sequence. The scanning model provides a framework for understanding these effects. The scanning mechanism also explains how small open reading frames near the 5' end of the mRNA can down-regulate translation. This constraint is sometimes abrogated by changing the structure of the mRNA, sometimes with clinical consequences. Examples are described. Some mistaken ideas about regulation of translation that have found their way into textbooks are pointed out and corrected.