Search

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell. The use of semipermeabilized cells has several advantages, in particular the avoidance of generating fragile spheroplasts and the opportunity of employing cells from a glucose-containing medium for APEX2 tagging. In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions to achieve high-resolution mapping of a subcellular yeast proteome.

Brefeldin A resistance factor 1 (Bfr1p) is a nonessential RNA-binding protein and multicopy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae. Deletion of BFR1 leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal missegregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent colocalization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA, which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1Δ strains show a strong reduction in Erg4p protein levels, most likely because of degradation of misfolded Erg4p. Polysome profiling of bfr1Δ or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation or increased ribosome loading. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA binding and cotranslational protein translocation into the ER and one in ploidy control or chromosome segregation.

The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). However, identification of the interacting proteome of a specific mRNA in vivo is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel RNA proximity biotinylation (RNA-BioID) method by tethering biotin ligase (BirA*) via MS2 coat protein (MCP) at the 3’-UTR of endogenous MS2 tagged β-actin mRNA (MBS) in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes upon serum-induced localization of the mRNA. Apart from the previously known interactors, we identified over 60 additional β-actin associated RBPs by RNA-BioID. Among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3’-UTR of β-actin mRNA, is essential for β-actin mRNA localization but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool to identify new mRNA interactors and to study the dynamic view of the interacting proteome of endogenous mRNAs in space and time.Significance statementTransport of specific mRNAs to defined sites in the cytoplasm allows local protein production and contributes to cell polarity, embryogenesis, and neuronal function. These localized mRNAs contain signals (zipcodes) that help directing them to their destination site. Zipcodes are recognized by RNA-binding proteins that, with the help of molecular motor proteins and supplementary factors, mediate mRNA trafficking. To identify all proteins assembling with a localized mRNA we advanced a proximity labeling method (BioID) by tethering a biotin ligase to the 3’ untranslated region of mRNA encoding the conserved beta-actin protein. We demonstrate that this method allows the identification of novel, functionally important proteins that are required for mRNA localization.

Proteins are positioned and act at defined subcellular locations. This is particularly important in eukaryotic cells that deliver proteins to membrane-bound organelles such as the endoplasmic reticulum (ER), mitochondria, or endosomes. It is axiomatic that organelle targeting depends mainly on polypeptide signals. However, recent results demonstrate that targeting elements within the encoding transcripts are essential for efficient protein localisation. Key readers of these elements are membrane-associated RNA-binding proteins (memRBPs) that orchestrate organelle-coupled translation. The translation products then either cross the membrane for organelle entry or hitchhike on organelle surfaces for complex assembly and co-transport. Understanding the interaction of protein- and RNA-based targeting signals is essential to decipher the molecular basis for mutant phenotypes in disease.

Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.

Summary: The RNA-binding protein Scp160p is the yeast homolog of the conserved vigilin protein family. These proteins influence a variety of nuclear and cytoplasmic functions. One of Scp160p’s reported roles is to increase translation elongation efficiency in a manner related to codon usage. Thus, it can affect translation speed and co-translational folding of nascent peptides. We used polyglutamine (polyQ) reporters to assess Scp160p’s effect on protein synthesis and observed that, in the absence of Scp160p, aggregation of polyQ is reduced and toxicity is abolished. We additionally took a proteomic approach and analyzed the impact of Scp160p on the aggregation of endogenous proteins under normal growth conditions. In the absence of Scp160p, aggregation of many Q/N-rich proteins was reduced. Because aggregation mediated by these regions can be important for the proteins’ functions, Scp160p may affect many processes via aggregation of Q/N-rich proteins. : Glutamine and asparagine (Q/N)-mediated protein aggregation can lead to neurodegenerative diseases but is also functionally important. Cheng et al. report a method to assess aggregation of proteins at the proteomic level and observe reduced aggregation of many endogenous Q/N-rich proteins in the absence of the yeast vigilin protein. Keywords: vigilin, polyglutamine, protein aggregation, codon usage, low complexity regions, SCP160

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA. ER targeting of at least two mRNAs, MID2 and SLG1/WSC1, requires KHD1 but is independent of their translation. Together, our results suggest interdependence of Khd1p and mRNA for their targeting to the ER and presents additional evidence for signal sequence-independent, RBP-mediated mRNA targeting.

mRNA localization is an essential mechanism of gene regulation and is required for processes such as stem-cell division, embryogenesis and neuronal plasticity. It is not known which features in the cis-acting mRNA localization elements (LEs) are specifically recognized by motor-containing transport complexes. To the best of our knowledge, no high-resolution structure is available for any LE in complex with its cognate protein complex. Using X-ray crystallography and complementary techniques, we carried out a detailed assessment of an LE of the ASH1 mRNA from yeast, its complex with its shuttling RNA-binding protein She2p, and its highly specific, cytoplasmic complex with She3p. Although the RNA alone formed a flexible stem loop, She2p binding induced marked conformational changes. However, only joining by the unstructured She3p resulted in specific RNA recognition. The notable RNA rearrangements and joint action of a globular and an unfolded RNA-binding protein offer unprecedented insights into the step-wise maturation of an mRNA-transport complex.

Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA's 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such proteins but suffer from the need for stable RNA-protein binding or direct contact of protein and RNA. To identify stably or transiently interacting proteins that directly or indirectly associated with the localization elements and the body of the mRNA, we developed an in vivo proximity labeling method we call RNA-BioID. In RNA-BioID, we tether a fusion of the BirA* biotin ligase and the MS2 coat protein (MCP) at the 3'-UTR of MS2-tagged β-actin mRNA in vivo. Exposing BirA* expressing cells to biotin in the media and induces biotinylation of β-actin mRNA-associated proteins that can be isolated with streptavidin beads. This technique allowed us to identify by mass spectrometry analysis the β-actin mRNA 3'-UTR-interacting proteome in fibroblasts. The protocol can be useful to identify the interacting proteome of any mRNA in mammalian cells.

Brefeldin A resistance factor 1 (Bfr1p) is a nonessential RNA-binding protein and multicopy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae. Deletion of BFR1 leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal missegregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent colocalization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA, which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1Δ strains show a strong reduction in Erg4p protein levels, most likely because of degradation of misfolded Erg4p. Polysome profiling of bfr1Δ or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation or increased ribosome loading. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA binding and cotranslational protein translocation into the ER and one in ploidy control or chromosome segregation.

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.

Posttranscriptional gene regulation is of great importance for a range of cellular functions. The active transport of transcripts and their local translation presents a mechanism important for embryo- and neurogenesis, neuronal plasticity, and stem cell differentiation. Impaired localization of transcripts result in erroneous embryogenesis, defective cell division, and neurological diseases. The DFG Research Unit FOR2333 has been established to mechanistically understand the processes of mRNA localization.

Eukaryotic cells rely on the precise determination of when and where proteins are synthesized. Spatiotemporal expression is supported by localization of mRNAs to specific subcellular sites and their subsequent local translation. This holds true for somatic cells as well as for oocytes and embryos. Most commonly, mRNA localization is achieved by active transport of the molecules along the actin or microtubule cytoskeleton. Key factors are molecular motors, adaptors, and RNA-binding proteins that recognize defined sequences or structures in cargo mRNAs. A deep understanding of this process has been gained from research on fungal model systems such as Saccharomyces cerevisiae and Ustilago maydis. Recent highlights of these studies are the following: (1) synergistic binding of two RNA-binding proteins is needed for high affinity recognition; (2) RNA sequences undergo profound structural rearrangements upon recognition; (3) mRNA transport is tightly linked to membrane trafficking; (4) mRNAs and ribosomes are transported on the cytoplasmic surface of endosomes; and (5) heteromeric protein complexes are, most likely, assembled co-translationally during endosomal transport. Thus, the study of simple fungal model organisms provides valuable insights into fundamental mechanisms of mRNA transport boosting the understanding of similar events in higher eukaryotes. WIREs RNA 2018, 9:e1453. doi: 10.1002/wrna.1453 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization.

The vigilin family of proteins is evolutionarily conserved from yeast to humans and characterized by the proteins’ 14 or 15 hnRNP K homology (KH) domains, typically associated with RNA-binding. Vigilin is the largest RNA-binding protein (RBP) in the KH domain-containing family and one of the largest RBP known to date. Since its identification 30 years ago, vigilin has been shown to bind over 700 mRNAs and has been associated with cancer progression and cardiovascular disease. We provide a brief historic overview of vigilin research and outline the proteins’ different functions, focusing on maintenance of genome ploidy, heterochromatin formation, RNA export, as well as regulation of translation, mRNA transport, and mRNA stability. The multitude of associated functions is reflected by the large number of identified interaction partners, ranging from tRNAs, mRNAs, ribosomes and ribosome-associated proteins, to histone methyltransferases and DNA-dependent protein kinases. Most of these partners bind to vigilin's carboxyterminus, and the two most C-terminal KH domains of the protein, KH13 and KH14, represent the main mRNA-binding interface. Since the nuclear functions of vigilins in particular are not conserved, we outline a model for the basal functions of vigilins, as well as those which were acquired during the transition from unicellular organisms to metazoa. WIREs RNA 2017, 8:e1448. doi: 10.1002/wrna.1448 For further resources related to this article, please visit the WIREs website.

Localization of mRNAs contributes to the generation and maintenance of cellular asymmetry in a wide range of organisms. In Saccharomyces cerevisiae, the so-called locasome complex with its core components Myo4p, She2p, and She3p localizes more than 30 mRNAs to the yeast bud tip. A significant fraction of these mRNAs encodes membrane or secreted proteins. Their localization requires, besides the locasome, a functional segregation apparatus of the cortical endoplasmic reticulum (ER), including the machinery that is involved in the movement of ER tubules into the bud. Colocalization of RNA-containing particles with these tubules suggests a coordinated transport of localized mRNAs and the cortical ER to the bud. Association of localized mRNAs to the ER requires the presence of the locasome component She2p. Here we report that She2p is not only an RNA-binding protein but can specifically bind to ER-derived membranes in a membrane curvature-dependent manner in vitro. Although it does not contain any known curvature recognizing motifs, the protein shows a binding preference for liposomes with a diameter resembling that of yeast ER tubules. In addition, membrane binding depends on tetramerization of She2p. In an in vivo membrane-tethering assay, She2p can target a viral peptide GFP fusion protein to the cortical ER, indicating that a fraction of She2p associates with the ER in vivo. Combining RNA- and membrane-binding features makes She2p an ideal coordinator of ER tubule and mRNA cotransport.

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1-3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co-localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2-tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co-translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo , suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.

RNA localization is a widespread mechanism that allows cells to spatially control protein function by determining their sites of synthesis. In embryos, localized mRNAs are involved in morphogen gradient formation or the asymmetric distribution of cell fate determinants. In somatic cell types, mRNA localization contributes to local assembly of protein complexes or facilitates protein targeting to organelles. Long-distance transport of specific mRNAs in plants allows coordination of developmental processes between different plant organs. In this review, we will discuss the biological significance of different patterns of mRNA localization.

Summary Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins [1, 2]. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, mRNA localization to the growing bud depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p [3]. These proteins mediate the localization of 23 mRNAs including ASH1 mRNA and mRNAs encoding membrane proteins [4]. In addition, Myo4p and She3p are required for segregation of cortical ER to the bud [5]. Here we show, with ASH1 mRNA as a model mRNA, that localizing messenger ribonucleoprotein (mRNP) particles comigrate with tubular ER structures to the bud, which requires the RNA binding protein She2p. Coordinated movement of the ASH1 mRNP with ER tubules but not their association with each other depends on Myo4p and She3p. Subcellular fractionation experiments demonstrate a cosegregation of ER and She2p, which is independent of Myo4p, She3p, or polysomes. Our findings suggest a novel model for mRNA localization that involves association of She2p and mRNPs with ER tubules and myosin-dependent cotransport of tubules and localized mRNPs.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

RNA localization and translational control are crucial for cellular fine-tuning of gene expression in space and time. A recent meeting in Tucson, Arizona pointed out mechanisms conserved across different species and cell types that contribute to the establishment of cell polarity and cell migration. Furthermore, it is becoming increasingly clear that these post-transcriptional control processes are relevant for various diseases.

In addition to its role in rRNA processing and ribosome assembly, the nucleolus plays a part in the assembly of non-ribosomal ribonucleoprotein particles (RNPs) that are destined for cytoplasmic RNA delivery. Recent evidence indicates that mammalian Staufen2, a brain-specific RNA-binding protein involved in RNA localization, can – at least transiently – enter the nucleolus. Therefore, the assembly of Staufen2 into transport-competent RNPs might occur in the nucleus before their export into the cytoplasm. This could provide new insights into the mechanisms of subcellular RNA localization.

Background: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. Results: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. Conclusions: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.

mRNA localization and localized translation is a common mechanism that contributes to cell polarity and cellular asymmetry. In metazoan, mRNA transport participates in embryonic axis determination and neuronal plasticity. Since the mRNA localization process and its molecular machinery are rather complex in higher eukaryotes, the unicellular yeast Saccharomyces cerevisiae has become an attractive model to study mRNA localization. Although the focus has so far been on the mechanism of ASH1 mRNA transport, it has become evident that mRNA localization also assists in protein sorting to organelles, as well as in polarity establishment and maintenance. A diversity of different pathways has been identified that targets mRNA to their destination site, ranging from motor protein-dependent trafficking of translationally silenced mRNAs to co-translational targeting, in which mRNAs hitch-hike to organelles on ribosomes during nascent polypeptide chain elongation. The presence of these diverse pathways in yeast allows a systemic analysis of the contribution of mRNA localization to the physiology of a cell.

Regulation of the localization of mRNAs and local translation are universal features in eukaryotes and contribute to cellular asymmetry and differentiation. In Saccharomyces cerevisiae, localization of mRNAs that encode membrane proteins requires the She protein machinery, including the RNA-binding protein She2p, as well as movement of the cortical endoplasmic reticulum (cER) to the yeast bud. In a screen for ER-specific proteins necessary for the directional transport of WSC2 and EAR1 mRNAs, we have identified enzymes that are involved in phospholipid metabolism. Loss of the phospholipid methyltransferase Cho2p, which showed the strongest impact on mRNA localization, disturbs mRNA localization, as well as ER morphology and segregation, owing to an increase in the amount of cellular phosphatidylethanolamine (PtdEtn). Mislocalized mRNPs containing She2p colocalize with aggregated cER structures, suggestive of the entrapment of mRNA and She2p by the elevated PtdEtn level. This was confirmed by the elevated binding of She2p to PtdEtn-containing liposomes. These findings underscore the importance of ER membrane integrity in mRNA transport.

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems. Important highlights are that RNA-binding proteins recognize cargo mRNA synergistically and that mRNAs are co-transported with membranous compartments such as the endoplasmic reticulum (ER) and endosomes. We further discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes, suggesting that this phenomenon is a common theme and not an exception restricted to fungi.

Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Cytoplasmic messenger RNA localization is a key post-transcriptional mechanism of establishing spatially restricted protein synthesis. The characterization of cis-acting signals within localized mRNAs, and the identification of trans-acting factors that recognize these signals, has opened avenues towards identifying the machinery and mechanisms involved in mRNA transport and localization.

RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences (‘zipcodes’) within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p’s C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA–motor protein adapters.

Asymmetric distribution of messenger RNAs is a widespread mechanism to localize synthesis of specific protein to distinct sites in the cell. Although not proven yet there is considerable evidence that mRNA localisation is an active process that depends on the activity of cytoskeletal motor proteins. To date, the only motor protein with a specific role in mRNA localisation is the budding yeast type V myosin Myo4p. Myo4p is required for the localisation of ASH1 mRNA, encoding a transcriptional repressor that is essential for differential expression of the HO gene and mating type switching in budding yeast. Mutations in Myo4p, in proteins of the actin cytoskeleton, and in four other specific genes, SHE2-SHE5 disrupt the daughter-specific localisation of ASH1 mRNA. In order to understand if Myo4p is directly participating in mRNA transport, we used in situ colocalisation and coprecipitation of Myo4p and ASH1 mRNA to test for their interaction. Our results indicate an association of Myo4p and ASH1 mRNA that depends on the activity of two other genes involved in ASH1 mRNA localisation, SHE2 and SHE3. This strongly suggests a direct role of Myo4p myosin as a transporter of localised mRNAs, convincingly supporting the concept of motor-protein based mRNA localisation.

It has become evident over the past years that a large fraction of messenger RNAs is tightly associated with the cytoskeleton. Whereas microtubules are involved in RNA-cytoskeletal association in large cells like oocytes, neurons, or oligodendrocytes, microfilaments play the major role in smaller somatic cell types. Association of RNA with cytoskeletal filaments clearly is required for mRNA transport, but also appears to be crucial for efficient protein synthesis. Recent data now shed light on how mRNAs attach to the cytoskeleton. Messenger RNA seems to interact with microtubules or microfilaments in the form of large ribonucleoprotein particles, which in some cases also contain components of the protein synthesis apparatus. Recently, a number of RNA binding proteins have been identified in flies, amphibians, and mammals that are essential for the interaction of mRNA with cytoskeletal filaments or with microtubule- or actin-associated proteins. Such proteins include heterologous ribonucleoproteins, which are also involved in nuclear export of RNA.

Cell divisions that produce progeny differing in their patterns of gene expression are key to the development of multicellular organisms. In the budding yeast Saccharomyces cerevisiae , mother cells but not daughter cells can switch mating type because they selectively express the HO endonuclease gene. This asymmetry is due to the preferential accumulation of an unstable transcriptional repressor protein, Ash1p, in daughter cell nuclei. Here it is shown that ASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin. A cis-acting element in the 3′-untranslated region of ASH1 mRNA is sufficient to localize a chimeric RNA to daughter cells. These results suggest that localization of mRNA may have been an early property of the eukaryotic lineage.

Asymmetric distribution of cytoplasmic proteins and messenger RNAs has been implicated in several instances of cell differentiation. Microtubules have been suggested to direct mRNA localization in Drosophila and Xenopus oocytes but motor proteins that might transport mRNAs have not yet been identified. Recent data imply that in Drosophila, Caenorhabditis elegans and budding yeast, proteins of the actin cytoskeleton, including unconventional myosins, play active roles in the segregation of differentiation factors and mRNAs.

Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs.

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1-3 protein machinery in Saccharomyces cerevisiae localizes30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co-localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2-tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co-translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.

At all steps from transcription to translation, RNA-binding proteins play important roles in determining mRNA function. Initially it was believed that for the vast majority of transcripts the role of RNA-binding proteins is limited to general functions such as splicing and translation. However, work from recent years showed that members of this class of proteins also recognize several mRNAs via cis-acting elements for their incorporation into large motor-containing particles. These particles are transported to distant subcellular sites, where they become subsequently translated. This process, called mRNA localization, occurs along microtubules or actin filaments, and involves kinesins, dyneins, as well as myosins. Although mRNA localization has been detected in a large number of organisms from fungi to humans, the underlying molecular machineries are not well understood. In this review we will outline general principles of mRNA localization and highlight three examples, for which a comparably large body of information is available. The first example is She2p/She3p-dependent localization of ASH1 mRNA in budding yeast. It is particularly well suited to highlight the interdependence between different steps of mRNA localization. The second example is Staufen-dependent localization of oskar mRNA in the Drosophila embryo, for which the importance of nuclear events for cytoplasmic localization and translational control has been clearly demonstrated. The third example summarizes Egalitarian/Bicaudal D-dependent mRNA transport events in the oocyte and embryo of Drosophila. We will highlight general themes and differences, point to similarities in other model systems, and raise open questions that might be answered in the coming years.

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

Yeast fibrillarin (NOP1) is an evolutionarily conserved, nucleolar protein necessary for multiple steps in ribosome biogenesis. Yeast mutants lacking a functional NOP1 gene can be complemented by human fibrillarin but are temperature sensitive for growth and impaired in pre-rRNA processing. In order to identify components which interact functionally with human fibrillarin in yeast, we isolated extragenic suppressors of this phenotype. One dominant suppressor, sof1-56, which is allele-specific for human fibrillarin and restores growth and pre-RNA processing at 35 degrees C, was cloned by in vivo complementation. The wild-type allele of SOF1 is essential for cell growth and encodes a novel 56 kDa protein. In its central domain, SOF1 contains a repeated sequence also found in beta-subunits of trimeric G-proteins and the splicing factor PRP4. A single amino acid exchange in the G beta-like repeat domain is responsible for the suppressing activity of sof1-56. Indirect immunofluorescence shows that SOF1 is located within the yeast nucleolus. Co-immunoprecipitation demonstrates the physical association of SOF1 with U3 small nucleolar RNA and NOP1. In vivo depletion of SOF1 leads to impaired pre-rRNA processing and inhibition of 18S rRNA production. Thus, SOF1 is a new component of the nucleolar rRNA processing machinery.

A conserved patch of amino acids in the globular tail of type V myosin binds She3p to localize ASH1 mRNA to the bud of dividing yeast cells., Type V myosin (MyoV)–dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2–tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail–lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.