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Marilyn Kozak

Publications and source records attributed to Marilyn Kozak.

8 recordsLinked to original sources
PubMed2006

Rethinking some mechanisms invoked to explain translational regulation in eukaryotes.

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.

Animals↗
PubMed2005

A second look at cellular mRNA sequences said to function as internal ribosome entry sites.

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.'

Humans↗
PubMed2005

Regulation of translation via mRNA structure in prokaryotes and eukaryotes.

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.

Amino Acid Sequence↗
PubMed2004

How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?

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.

3' Untranslated Regions↗
PubMed2003

Alternative ways to think about mRNA sequences and proteins that appear to promote internal initiation of translation.

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.

Animals↗
PubMed2002

Pushing the limits of the scanning mechanism for initiation of 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.

Animals↗
PubMed2002

Emerging links between initiation of translation and human diseases.

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.

Cell Transformation, Neoplastic↗