[mRNA Targeting, Transport and Local Translation in Eukaryotic Cells: From the Classical View to a Diversity of New Concepts].

Spatial organization of protein biosynthesis in the eukaryotic cell has been studied for more than fifty years, thus many facts have already been included in textbooks. According to the classical view, mRNA transcripts encoding secreted and transmembrane proteins are translated by ribosomes associated with endoplasmic reticulum membranes, while soluble cytoplasmic proteins are synthesized on free polysomes. However, in the last few years, new data has emerged, revealing selective translation of mRNA on mitochondria and plastids, in proximity to peroxisomes and endosomes, in various granules and at the cytoskeleton (actin network, vimentin intermediate filaments, microtubules and centrosomes). There are also long-standing debates about the possibility of protein synthesis in the nucleus. Localized translation can be determined by targeting signals in the synthesized protein, nucleotide sequences in the mRNA itself, or both. With RNA-binding proteins, many transcripts can be assembled into specific RNA condensates and form RNP particles, which may be transported by molecular motors to the sites of active translation, form granules and provoke liquid-liquid phase separation in the cytoplasm, both under normal conditions and during cell stress. The translation of some mRNAs occurs in specialized "translation factories," assemblysomes, transperons and other structures necessary for the correct folding of proteins, interaction with functional partners and formation of oligomeric complexes. Intracellular localization of mRNA has a significant impact on the efficiency of its translation and presumably determines its response to cellular stress. Compartmentalization of mRNAs and the translation machinery also plays an important role in viral infections. Many viruses provoke the formation of specific intracellular structures, virus factories, for the production of their proteins. Here we review the current concepts of the molecular mechanisms of transport, selective localization and local translation of cellular and viral mRNAs, their effects on protein targeting and topogenesis, and on the regulation of protein biosynthesis in different compartments of the eukaryotic cell. Special attention is paid to new systems biology approaches, providing new cues to the study of localized translation.

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis.

Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.

Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling.

The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

Upstream open reading frames regulate translation of the long isoform of SLAMF1 mRNA that encodes costimulatory receptor CD150.

More than 40% of human genes contain upstream open reading frames (uORF) in their 5'-untranslated regions (5'-UTRs) and at the same time express at least one truncated mRNA isoform containing no uORF. We studied translational regulation by four uORFs found in the 5'-UTR of full-length mRNA for SLAMF1, the gene encoding CD150 membrane protein. CD150 is a member of the CD2 superfamily, a costimulatory lymphocyte receptor, a receptor for measles virus, and a microbial sensor on macrophages. The SLAMF1 gene produces at least two mRNA isoforms that differ in their 5'-UTRs. In the long isoform of the SLAMF1 mRNA that harbors four uORFs in the 5'-UTR, the stop codon of uORF4 overlaps with the AUG codon of the main ORF forming a potential termination-reinitiation site UGAUG, while uORF2 and uORF3 start codons flank a sequence identical to Motif 1 from the TURBS regulatory element. TURBS was shown to be required for a coupled termination-reinitiation event during translation of polycistronic RNAs of some viruses. In a model cell system, reporter mRNA based on the 5'-UTR of SLAMF1 short isoform, which lacks any uORF, is translated 5-6 times more efficiently than the mRNA with 5'-UTR from the long isoform. Nucleotide substitutions disrupting start codons in either uORF2-4 result in significant increase in translation efficiency, while substitution of two nucleotides in TURBS Motif 1 leads to a 2-fold decrease in activity. These data suggest that TURBS-like elements can serve for translation control of certain cellular mRNAs containing uORFs.

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.

[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.

[Minor secondary-structure variation in the 5'-untranslated region of the beta-globin mRNA changes the concentration requirements for eIF2]. / Neznachitel'nye variatsii vtorichnoi struktury 5'-netransliruemoi oblasti mRNK beta-globina izmeniaiut trebovaniia k kontsentratsii faktora initsiatsii eIF2.

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure elements in the 5'-untranslated region (5'-UTR) of the beta-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at its low concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF3B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the beta-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the complex of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR.