A deterministic model for non-monotone relationship between translation of upstream and downstream open reading frames.

Totally asymmetric simple exclusion process (TASEP) modelling was shown to offer a parsimonious explanation for the experimentally confirmed ability of a single upstream open reading frames (uORFs) to upregulate downstream translation during the integrated stress response. As revealed by numerical simulations, the model predicts that reducing the density of scanning ribosomes upstream of certain uORFs increases the flow of ribosomes downstream. To gain a better insight into the mechanism which ensures the non-monotone relation between the upstream and downstream flows, in this work, we propose a phenomenological deterministic model approximating the TASEP model of the translation process. We establish the existence of a stationary solution featuring the decreasing density along the uORF for the deterministic model. Further, we find an explicit non-monotone relation between the upstream ribosome density and the downstream flow for the stationary solution in the limit of increasing uORF length and increasingly leaky initiation. The stationary distribution of the TASEP model, the stationary solution of the deterministic model and the explicit limit are compared numerically.

[Determination of the Minimal Fragment of the Poliovirus IRES Necessary for the Formation of a Specific Complex with the Human Glycyl-tRNA Synthetase].

Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charge a tRNA molecule with a cognate amino acid required for protein synthesis. Glycyl-tRNA synthetase is one of the most interesting aminoacyl-tRNA synthetases due to its structure variability and functional features in the different organisms. It was shown recently that human glycyl-tRNA synthetase is a regulator of translational initiation of poliovirus mRNA. Details of this process and its mechanism still remain unknown. While exploring this stage of poliovirus functioning we have studied the interaction of the cytoplasmic form of human glycyl-tRNA synthetase and its domains with the fragments of the poliovirus IRES element. As a result, we have identified the minimal fragment of viral mRNA with which glycyl-tRNA synthetase fully interacts and estimated the contribution of some domains to the interaction of glycyl-tRNA synthetase with RNA.

Cap-independent translation initiation of apaf-1 mRNA based on a scanning mechanism is determined by some features of the secondary structure of its 5' untranslated region.

We have earlier shown that the 5'-untranslated region (5' UTR) of the mRNA coding for activation factor of apoptotic peptidase 1 (Apaf-1) can direct translation in vivo by strictly 5' end-dependent way even in the absence of m(7)G-cap. Dependence of translational efficiency on the cap availability for this mRNA turned out to be relatively low. In this study we demonstrate that this surprising phenomenon is determined the 5'-proximal part (domains I and II) of highly structured Apaf-1 5' UTR. Remarkably, domain II by itself was able to reduce dependence of the mRNA on the cap on its transferring to a short 5' UTR derived from a standard vector. We suggest that the low cap-dependence inherent to some cellular mRNAs may have an important physiological significance under those stress conditions when the function of cap-binding factor eIF4E is impaired.

Cap- and IRES-independent scanning mechanism of translation initiation as an alternative to the concept of cellular IRESs.

During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However, many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes a set of control experiments, which should be performed before concluding that a 5' UTR of eukaryotic mRNA does contain an IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing to the existence of a cap- and IRES-independent scanning mechanism of translation initiation based on utilization of special RNA structures called cap-independent translational enhancers (CITE). We describe this mechanism and suggest it as an alternative to the concept of cellular IRESs.

[Efficient cap-dependent in vitro and in vivo translation of mammalian mRNAs with long and highly structured 5'-untranslated regions].

According to generally accepted scanning model proposed by M. Kozak, the secondary structure of 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs can only cause an inhibitory effect on the translation initiation since it would counteract migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. Thus, the existence of efficiently translatable mRNAs with long and highly structured 5'-UTRs is not compatible with the cap-dependent scanning mechanism. It is expected that such mRNAs should use alternative ways of translation initiation to be efficiently translated, first of all the mechanism of the internal ribosome entry mediated by special RNA structures called IRESes (for Internal Ribosome Entry Sites), which have been proposed to reside within their 5'-UTRs. In this paper, it is shown that this point of view is not correct and most probably based on experiments of mRNA translation in rabbit reticulocyte lysate. This cell free system does not reflect correctly the ratio of translation efficiencies of various mRNAs which is observed in the living cell. Using five different mRNAs of similar design which possess either relatively short leaders of cellular mRNAs (beta-globin and beta-actin mRNAs) or long and highly structured 5'-UTRs (c-myc, LINE-1, Apaf-1 mRNAs), we show that the translation activities of all tested 5'-UTRs are comparable, both in transfected cells and in a whole cytoplasmic extract of cultivated cells. This activity is strongly dependent on the presence of the cap at their 5'-ends.

[A key role of the internal region of the 5'-untranslated region in the human L1 retrotransposon transcription activity].

The long 5-untranslated region (5'-UTR) of the human retrotransposon L1 harbors a unique internal promoter which ensures new copies of this mobile element to be much less dependent on an integration site at the level of transcription. The mechanism of this promoter's action still remains unclear, but due to some early studies the opinion has been -formed that the most important part for its function ("minimal promoter") is the first 100-150 nts of the 5'-UTR. In this paper we show that activity of the "minimal promoter" is rather poor in comparison with the entire 5'-UTR. The absolutely crucial part which is indispensable for the effective transcription is the internal region of the 5'-UTR (+390...+662) containing multiple binding sites for various transcription factors. This region may be considered as a transcriptional enhancer. Deletion of this segment leads to a dramatic lost of transcription level irrespectively of cell type, while deletion of the first 100 nt decreases the transcription efficiency no more than 1.5 to 2-fold. Thus, the organization of the L1 regulatory region may be much more similar to that of well-studied invertebrate LINE elements than it was thought before. Also we suggest a possible existence of an alternative sense promoter within the internal part of the L1 5'-UTR driving the synthesis of a 5'-truncated mRNA of the retrotransposon.

[Similar features in mechanisms of translation initiation of mRNAs in eukaryotic and prokaryotic systems].

Using as examples non-canonical features of translation initiation for some bacterial and mammalian mRNAs with unusual 5'- untranslated regions (5'-UTR) or lacking these regions (leaderless mRNAs), the authors of this review discuss similarities in mechanisms of translation initiation on prokaryotic and eukaryotic ribosomes.

[Adequate system for investigation of translation initiation of the human retrotransposon L1 mRNA in vitro].

Retrotransposon L1 codes for a unique dicistronic mRNA which serves both a transposition intermediate and a template for the synthesis of two proteins of this mobile element. According to preliminary data, the translation initiation of both cistrons of L1 occurs by non-canonical mechanisms. When translating the L1 mRNA in rabbit reticulocyte lysate (RRL), a standard system routinely used by many researchers to study mechanisms of translation initiation in eukaryotes, we observed along with expected products a number of polypeptides resulted from aberrant initiation at internal AUG codons. Such products are absent on translation of L1 mRNA in vivo. Addition to the system of a cytoplasmic extract from HeLa cells resulted in disappearance of these abberant products whereas the efficiency of translation of the first cistron remained unchanged. The level of translation of the second cistron became significantly lower. This also made the picture closer to that observed in vivo. These and other experiments allowed us to clearly demonstrate that the new combined cell-free system is much more adequate to study mechanisms of translation initiation than a regular RRL.