mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.
mRNA localization; She2p; She3p; ASH1 mRNA; Translational control; Myosin
Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3′UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.
Spatial control of mRNA translation is a well established mechanism for generating cellular asymmetries and for functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis . Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation . Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Mutations that abolish nos localization prevent abdominal development whereas de-repression of unlocalized nos mRNA suppresses head/thorax development, indicating that spatial regulation of nos is essential for anterior-posterior patterning [3, 5]. The observation that both loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons suggests that nos might also be regulated in these larval sensory neurons . Here we show that localization and translational control of nos mRNA are essential for late stages of da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons, but suggests a different transport mechanism. The need to target nos mRNA to the processes of da neurons may reflect a requirement for Nos protein in controlling translation locally within dendrites.
In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5′–3′ mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.
Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.
Axons and their growth cones are specialized neuronal sub-compartments that possess translation machinery and have distinct messenger RNAs (mRNAs). Several classes of mRNAs have been identified using candidate-based, as well as unbiased genome-wide-based approaches. Axonal mRNA localization serves to regulate spatially the protein synthesis; thereby, providing axons with a high degree of functional autonomy from the soma during axon pathfinding. Importantly, de novo protein synthesis in navigating axonal growth cones is necessary for chemotropic responses to various axon guidance cues. This chapter discusses the molecular components involved in regulating axonal mRNA trafficking, targeting, and translation, and focuses on RNA binding proteins (RNBPs) and microRNAs. The functional significance of local mRNA translation in the directional response of growth cones to a gradient is highlighted along with the downstream signaling events that mediate local protein synthesis. The view that emerges is that local translation is tightly coupled to extracellular cues, enabling growth cones to respond to new signals with exquisite adaptability and spatiotemporal control.
Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40–220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3′-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA–protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.
Messenger RNAs that are associated with each of the five Puf RNA-binding proteins can be grouped according to function and localization, suggesting that tagging of transcripts by specific RNA-binding proteins coordinates the control of their localization, translation, and decay
Though posttranscriptional regulation is important for early embryogenesis, little is understood regarding control of mRNA decay during development. Previous work defined two major pathways by which normal transcripts are degraded in eukaryotes. However it is not known which pathways are key in mRNA decay during early patterning or whether developmental transcripts are turned over via specific pathways. Here we show that Caenorhabditis elegans Dcp2 is localized to distinct foci during embryogenesis, reminiscent of P-bodies, the sites of mRNA degradation in yeast and mammals. However the decapping enzyme of the 3′ to 5′ transcript decay system (DcpS) localizes throughout the cytoplasm, suggesting this degradation pathway is not highly organized. In addition we find that Dcp2 is localized to P-granules, showing that Dcp2 is stored and/or active in these structures. However RNAi of these decapping enzymes has no obvious effect on embryogenesis. In contrast we find that nuclear cap binding proteins (CBP-20 and 80), eIF4G, and PAB-1 are absolutely required for development. Together our data provides further evidence that pathways of general mRNA metabolism can be remarkably organized during development, with two different decapping enzymes localized in distinct cytoplasmic domains.
After export from the nucleus it turns out that all mRNAs are not treated equally. Not only is mRNA subject to translation, but through RNA binding proteins and other trans-acting factors, eukaryotic cells interpret codes for spatial sorting within the mRNA sequence. These codes instruct the cytoskeleton and translation apparatus to make decisions about where to transport and when to translate the intended protein product. Signaling pathways decode extra-cellular cues and can modify transport and translation factors in the appropriate cytoplasmic space to achieve translation locally. Identifying regulatory sites on transport factors as well as novel physiological functions for well known translation factors have provided significant advances in how spatially controlled translation impacts cell function.
The importance of mRNA localization and localized protein synthesis to spatially modulate protein levels in distinct subcellular domains has increasingly been recognized in recent years. Axonal and dendritic processes of neurons represent separate functional domains of the cell that have shown the capacity to autonomously respond to extracellular stimuli through localized protein synthesis. With the vast distance often separating distal axons and dendrites from the neuronal cell body, these processes have provided an appealing and useful model system to study the mechanisms that drive mRNA localization and regulate localized mRNA translation. Here, we discuss the methodologies that have been used to isolate neuronal processes to purity, and provide an in-depth method for using a modified Boyden chamber to isolate axons from adult dorsal root ganglion neurons for analyses of axonal mRNA content. We further show how this method can be utilized to identify specific mRNAs whose transport into axons is altered in response to extracellular stimuli, providing a means to begin to understand how axonal protein synthesis contributes to the proper function of the neuron.
mRNA localization; RNA transport; axonal protein synthesis; local translation
Restriction of proteins to discrete subcellular regions is a common mechanism to establish cellular asymmetries and depends on a coordinated program of mRNA localization and translation control. Many processes from the budding of a yeast to the establishment of metazoan embryonic axes and the migration of human neurons, depend on this type of cell polarization. How factors controlling transport and translation assemble to regulate at the same time the movement and translation of transported mRNAs, and whether these mechanisms are conserved across kingdoms is not yet entirely understood. In this review we will focus on some of the best characterized examples of mRNA transport machineries, the “yeast locasome” as an example of RNA transport and translation control in unicellular eukaryotes, and on the Drosophila Bic-D/Egl/Dyn RNA localization machinery as an example of RNA transport in higher eukaryotes. This focus is motivated by the relatively advanced knowledge about the proteins that connect the localizing mRNAs to the transport motors and the many well studied proteins involved in translational control of specific transcripts that are moved by these machineries. We will also discuss whether the core of these RNA transport machineries and factors regulating mRNA localization and translation are conserved across eukaryotes.
Localization of mRNAs to dendrites and local protein synthesis afford spatial and temporal regulation of gene expression and endow synapses with the capacity to autonomously alter their structure and function. Emerging evidence indicates that RNA binding proteins, ribosomes, translation factors and mRNAs encoding proteins critical to synaptic structure and function localize to neuronal processes. RNAs are transported into dendrites in a translationally quiescent state where they are activated by synaptic stimuli. Two RNA binding proteins that regulate dendritic RNA delivery and translational repression are cytoplasmic polyadenylation element binding protein and fragile X mental retardation protein (FMRP). The fragile X syndrome (FXS) is the most common known genetic cause of autism and is characterized by the loss of FMRP. Hallmark features of the FXS include dysregulation of spine morphogenesis and exaggerated metabotropic glutamate receptor-dependent long term depression, a cellular substrate of learning and memory. Current research focuses on mechanisms whereby mRNAs are transported in a translationally repressed state from soma to distal process and are activated at synaptic sites in response to synaptic signals.
synaptic plasticity; synaptic signaling; translational control; fragile X syndrome; FMRP; CPEB; cytoplasmic polyadenylation
mRNAs can be targeted to specific neuronal subcellular domains, which enables rapid changes in the local proteome through local translation. This mRNA-based mechanism links extrinsic signals to spatially restricted cellular responses and can mediate stimulus-driven adaptive responses such as dendritic plasticity. Local mRNA translation also occurs in growing axons where it can mediate directional responses to guidance signals. Recent profiling studies have revealed that both growing and mature axons possess surprisingly complex and dynamic transcriptomes, thereby suggesting that axonal mRNA localization is highly regulated and has a role in a broad range of processes, a view that is increasingly being supported by new experimental evidence. Here, we review current knowledge on the roles and regulatory mechanisms of axonal mRNA translation and discuss emerging links to axon guidance, survival, regeneration and neurological disorders.
Neurons are exquisitely polarized cells that extend intricate axonal and dendritic arbors. Developmental cues guide axons and dendrites into circuits by inducing rapid changes in local protein expression and cytoskeleton structure. Neurons can transduce these signals through local mRNA regulation. Here, we review the latest insights regarding post-transcriptional control of gene expression through mRNA transport and local protein synthesis in developing neurons. We focus on local mRNA regulation during axon growth and guidance, dendrite morphogenesis, and synapse formation and refinement. Dysregulated mRNA transport and translation in neurological disorders are also discussed. The collection of molecules and mechanisms reviewed includes sequence-specific RNA binding proteins, microtubule motors and adaptors, microRNAs, translation initiation factors, and the receptor-mediated signaling that modulates these molecules.
Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.
The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the Saccharomyces cerevisiae Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5′ to 3′ mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5′ to 3′ mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5′ to 3′ decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5′ to 3′ exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5′ to 3′ mRNA decay is essential for the packaging of Ty1 RNA in VLPs.
Intracellular messenger RNA (mRNA) traffic and translation must be highly regulated, both temporally and spatially, within eukaryotic cells to support the complex functional partitioning. This capacity is essential in neurons because it provides a mechanism for rapid input-restricted activity-dependent protein synthesis in individual dendritic spines. While this feature is thought to be important for synaptic plasticity, the structures and mechanisms that support this capability are largely unknown. Certainly specialized RNA binding proteins and binding elements in the 3′ untranslated region (UTR) of translationally regulated mRNA are important, but the subtlety and complexity of this system suggests that an intermediate “specificity” component is also involved. Small non-coding microRNA (miRNA) are essential for CNS development and may fulfill this role by acting as the guide strand for mediating complex patterns of post-transcriptional regulation. In this review we examine post-synaptic gene regulation, mRNA trafficking and the emerging role of post-transcriptional gene silencing in synaptic plasticity.
MicroRNA; Gene silencing; Synaptic plasticity; Dendritic spines; Memory
In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.
Local regulation of protein synthesis in neurons has emerged as a leading research focus due to its importance in synaptic plasticity and neurological diseases. The complexity of neuronal sub-cellular domains and their distance from the soma demand local spatial and temporal control of protein synthesis. Synthesis of many synaptic proteins, such as GluR and PSD-95, is under local control. mRNA binding proteins (RBPs), such as FMRP, function as key regulators of local RNA translation, and the mTORC1 pathway acts as a primary signaling cascade for regulation of these proteins. Much of the regulation occurs through structures termed RNA granules, which are based on reversible aggregation of the RBPs, some of which have aggregation prone domains with sequence features similar to yeast prion proteins. Mutations in many of these RBPs are associated with neurologic diseases, including FMRP in Fragile X syndrome, TDP-43, FUS, angiogenin and ataxin-2 in amyotrophic lateral sclerosis, ataxin-2 in spinocerebellar ataxia, and SMN in spinal muscular atrophy.
Protein synthesis; RNA translation; Synaptic efficacy; transport; RNA granule; Stress Granule; amyotrophic lateral sclerosis; fragile X syndrome; spinal muscular atrophy; spinocerebellar ataxia
MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.
MicroRNA; Neuronal development; Synapse; Learning and memory; Neurological diseases
piRNAs and Piwi proteins have been implicated in transposon control and are linked to transposon methylation in mammals. Here, we examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in Dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells.
Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.
The assembly of functional neural circuits is critical for complex thoughts, behavior and general brain function. Precise construction of neural circuits requires orderly transition of sequential events from axon outgrowth, pathfinding, branching, to synaptogenesis. Each of these steps is required to be tightly regulated in order to achieve meticulous formation of neuronal connections. MicroRNAs (miRNAs), which silence gene expression post-transcriptionally via either inhibition of translation or destabilization of messenger RNAs, have emerged as key regulators of neuronal connectivity. The expression of miRNAs in neurons is often temporally and spatially regulated, providing critical timing and local mechanisms that prime neuronal growth cones for dynamic responses to extrinsic cues. Here we summarize recent findings of miRNA regulation of neuronal connectivity in a variety of experimental platforms.
miRNAs; neuronal connectivity; axon pathfinding; axon branching; timing mechanisms; temporal regulation; heterochronic miRNAs; axon outgrowth
mRNA localization and regulated translation provide a means of spatially restricting gene expression within each of the thousands of subcellular compartments made by a neuron, thereby vastly increasing the computational capacity of the brain. Recent studies reveal that local translation is regulated by stimuli that trigger neurite outgrowth/collapse, axon guidance, synapse formation, pruning, activity-dependent synaptic plasticity, and injury induced axonal regeneration. Impairments in the local regulation of translation result in aberrant signaling, physiology, and morphology of neurons, and are linked to neurological disorders. This review highlights current advances in understanding how mRNAs are translationally repressed during transport and how local translation is activated by stimuli. We address the function of local translation in the context of fragile X mental retardation.
Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5′-to-3′ exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.