mRNA Targeting, Transport and Local Translation in Eukaryotic Cells: From the Classical View to a Diversity of New Concepts.
TL;DR: A review of 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 can be found in this paper.
Abstract: 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.
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TL;DR: In this article, the authors propose to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment, such as Internal Ribosome Entry Sites (IRESs), which make translation independent of the free 5′ end, or cap-independent translational enhancers (CITEs) which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs).
Abstract: Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5′ end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5′ end, even if located in 3′ untranslated regions (3′ UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular “translation factories”, which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles’ heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.
14 citations
14 Feb 2022
TL;DR: This review focuses on emerging concepts regarding the functional outcomes of mRNA targeting in the cytoplasm of highly polarized cells and regulatory mechanisms controlling these events, with an emphasis on the role of cell mechanics and the organization of the cytoskeleton.
Abstract: Spatial segregation of mRNAs in the cytoplasm of cells is a well‐known biological phenomenon that is widely observed in diverse species spanning different kingdoms of life. In mammalian cells, localization of mRNAs has been documented and studied quite extensively in highly polarized cells, most notably in neurons, where localized mRNAs function to direct protein production at sites that are quite distant from the soma. Recent studies have strikingly revealed that a large proportion of the cellular transcriptome exhibits polarized distributions even in cells that lack an obvious need for long‐range transport, such as fibroblasts or epithelial cells. This review focuses on emerging concepts regarding the functional outcomes of mRNA targeting in the cytoplasm of such cells. We also discuss regulatory mechanisms controlling these events, with an emphasis on the role of cell mechanics and the organization of the cytoskeleton.
12 citations
TL;DR: In this article, the authors suggest cellular circadian timing primarily functions to maintain proteome homeostasis rather than perturb it, and that optimal bioenergetic efficiency results from coupled rhythms in mammalian target of rapamycin complex activity, protein synthesis/turnover, ion transport and protein sequestration, which drive facilitatory rhythms in metabolic flux and substrate utilisation.
12 citations
TL;DR: In this article, the authors provide an overview of the molecular functions of translation factors and ribosomal proteins in the cell nucleus, as well as the role of these components in various nuclear processes, particularly those associated with genome integrity control and the nuclear stages of gene expression.
Abstract: Components of the translation apparatus, including ribosomal proteins, have been found in cell nuclei in various organisms. Components of the translation apparatus are involved in various nuclear processes, particularly those associated with genome integrity control and the nuclear stages of gene expression, such as transcription, mRNA processing, and mRNA export. Components of the translation apparatus control intranuclear trafficking; the nuclear import and export of RNA and proteins; and regulate the activity, stability, and functional recruitment of nuclear proteins. The nuclear translocation of these components is often involved in the cell response to stimulation and stress, in addition to playing critical roles in oncogenesis and viral infection. Many components of the translation apparatus are moonlighting proteins, involved in integral cell stress response and coupling of gene expression subprocesses. Thus, this phenomenon represents a significant interest for both basic and applied molecular biology. Here, we provide an overview of the current data regarding the molecular functions of translation factors and ribosomal proteins in the cell nucleus.
6 citations
TL;DR: The Arabidopsis thaliana Friendly (FMT) protein is shown to be a cytosolic RNA binding protein that associates with cytOSolic ribosomes at the surface of mitochondria, indicating that FMT participates in the localization of mRNAs and their translation at thesurface of mitochondia.
Abstract: The spatial organization of protein synthesis in the eukaryotic cell is essential for maintaining the integrity of the proteome and the functioning of the cell. Translation on free polysomes or on ribosomes associated with the endoplasmic reticulum has been studied for a long time. More recent data have revealed selective translation of mRNAs in other compartments, in particular at the surface of mitochondria. Although these processes have been described in many organisms, in particular in plants, the mRNA targeting and localized translation mechanisms remain poorly understood. Here, the Arabidopsis thaliana Friendly (FMT) protein is shown to be a cytosolic RNA binding protein that associates with cytosolic ribosomes at the surface of mitochondria. Its knockdown delays seedling development and causes mitochondrial clustering. The mutation also disrupts the mitochondrial proteome and the localization of nuclear transcripts on the surface of mitochondria. These data indicate that FMT participates in the localization of mRNAs and their translation at the surface of mitochondria.
3 citations
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TL;DR: This work has shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates and has proposed a physical framework for this organizing principle.
Abstract: In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.
3,294 citations
TL;DR: In this article, it was shown that in vitro completion of these nascent light chains resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chain, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same moll wt, as the precursor of the light chain.
Abstract: Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.
2,571 citations
TL;DR: This report reports multiplexed error-robust FISH (MERFISH), a single-molecule imaging method that allows thousands of RNA species to be imaged in single cells by using combinatorial FISH labeling with encoding schemes capable of detecting and/or correcting errors.
Abstract: INTRODUCTION: The copy number and in- tracellular localization of RNA are important regulators of gene expression. Measurement of these properties at the transcriptome scale in single cells will give answers to many ques- tions related to gene expression and regulation. Single-molecule RNA imaging approaches, such as single-molecule fluorescence in situ hybrid- ization(smFISH), are powerful toolsforcount- ing and mappingRNA; however, the number of RNA species that can be simultaneously im- aged in individual cells has been limited. This makes it challenging to perform transcriptomic analysis of single cells in a spatially resolved manner. Here, we report multiplexed error- robust FISH (MERFISH), a single-molecule im- aging method that allows thousands of RNA species to be imaged in single cells by using combinatorial FISH labeling with encoding schemes capable of detecting and/or correct- ing errors. RATIONALE: We labeled each cellular RNA with a set of encoding probes, which contain targeting sequences that bind the RNA and readout sequences that bind fluorescently la- beled readout probes. Each RNA species is encodedwithaparticular combinationofread- out sequences. We used successive rounds of hybridization and imaging, each with a differ- ent readout probe, to identify the readout se- quences bound to each RNA and to decode the RNA. In principle, combinatorial labeling al- lows the number of detectable RNA species to
1,576 citations
TL;DR: Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms.
1,089 citations
TL;DR: A high-resolution fluorescent in situ hybridization procedure was developed and employed to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis, indicating major roles for mRNA localization in nucleating localized cellular machineries.
942 citations