A system-wide quantitative map of RNA and protein subcellular localisation dynamics (original) (raw)
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Atlas of Subcellular RNA Localization Revealed by APEX-seq
We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
Subcellular mRNA localisation at a glance
Journal of cell science, 2014
mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, ...
Diverse ribonucleoprotein complexes control messenger RNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-Seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS) provides new insights into the organization of translation initiation complexes on active mRNAs, as well as exposing unanticipated complexity in stress granule composition, and provides a powerful and general approach to explore the spatial environment of macromolecules. .
Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways
FEBS letters, 2018
The asymmetric subcellular distribution of RNA molecules from their sites of transcription to specific compartments of the cell is an important aspect of post-transcriptional gene regulation. This involves the interplay of intrinsic cis-regulatory elements within the RNA molecules with trans-acting RNA-binding proteins and associated factors. Together, these interactions dictate the intracellular localization route of RNAs, whose downstream impacts have wide-ranging implications in cellular physiology. In this review, we examine the mechanisms underlying RNA localization and discuss their biological significance. We also review the growing body of evidence pointing to aberrant RNA localization pathways in the development and progression of diseases.
Genomic analysis of RNA localization
RNA biology, 2014
The localization of mRNAs to specific subcellular sites is widespread, allowing cells to spatially restrict and regulate protein production, and playing important roles in development and cellular physiology. This process has been studied in mechanistic detail for several RNAs. However, the generality or specificity of RNA localization systems and mechanisms that impact the many thousands of localized mRNAs has been difficult to assess. In this review, we discuss the current state of the field in determining which RNAs localize, which RNA sequences mediate localization, the protein factors involved, and the biological implications of localization. For each question, we examine prominent systems and techniques that are used to study individual messages, highlight recent genome-wide studies of RNA localization, and discuss the potential for adapting other high-throughput approaches to the study of localization.
RNA localization in yeast: moving towards a mechanism
Biology of the Cell, 2005
RNA localization is a widely utilized strategy employed by cells to spatially restrict protein function. In Saccharomyces cerevisiae asymmetric sorting of mRNA to the bud has been reported for at least 24 mRNAs. The mechanism by which the mRNAs are trafficked to the bud, illustrated by ASH1 mRNA, involves recognition of cis-acting localization elements present in the mRNA by the RNA-binding protein, She2p. The She2p/mRNA complex subsequently associates with the myosin motor protein, Myo4p, through an adapter, She3p. This ribonucleoprotein complex is transported to the distal tip of the bud along polarized actin cables. While the mechanism by which ASH1 mRNA is anchored at the bud tip is unknown, current data point to a role for translation in this process, and the rate of translation of Ash1p during the transport phase is regulated by the cis-acting localization elements. Subcellular sorting of mRNA in yeast is not limited to the bud; certain mRNAs corresponding to nuclear-encoded mitochondrial proteins are specifically sorted to the proximity of mitochondria. Analogous to ASH1 mRNA localization, mitochondrial sorting requires cis-acting elements present in the mRNA, though trans-acting factors involved with this process remain to be identified. This review aims to discuss mechanistic details of mRNA localization in S. cerevisiae.
Proceedings of the National Academy of Sciences of the United States of America, 2006
Argonaute proteins associate with microRNAs (miRNAs) that bind mRNAs through partial base-pairings to primarily repress translation in animals. A fraction of Argonaute proteins and miRNAs biochemically cosediment with polyribosomes, yet another fraction paradoxically accumulates in ribosome-free processing bodies (PBs) in the cytoplasm. In this report, we give a quantitative account of the Argonaute protein localization and dynamics in living cells in different cellular states. We find that the majority of Argonaute is distributed diffusely in the cytoplasm, and, when cells are subjected to stress, Argonaute proteins accumulate to newly assembled structures known as stress granules (SGs) in addition to PBs. Argonaute proteins displayed distinct kinetics at different structures: exchange faster at SGs and much slower at PBs. Further, miRNAs are required for the Argonaute protein localization to SGs but not PBs. These quantitative kinetic data provide insights into miRNA-mediated repression.
Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality
Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA processing enzymes, termed processing bodies (PBs). Here, we track the dynamic localization of individual miRNAs, mRNAs and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and positioning of cis-regulatory elements significantly impact PB-localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches we show that phase separation into large PBs attenuates mRNA silencing, suggesting that physiological mRNA turnover predominantly occurs outside of PBs. Instead, our data support a role for PBs in sequestering unused miRNAs to regulate their surv...
Spatial regulation of translation through RNA localization
F1000 Biology Reports, 2012
RNA localization is a mechanism to post-transcriptionally regulate gene expression. Eukaryotic organisms ranging from fungi to mammals localize mRNAs to spatially restrict synthesis of specific proteins to distinct regions of the cytoplasm. In this review, we provide a general summary of RNA localization pathways in Saccharomyces cerevisiae, Xenopus, Drosophila and mammalian neurons.
mRNA Localization: Gene Expression in the Spatial Dimension
Cell, 2009
The localization of mRNAs to subcellular compartments provides a mechanism for regulating gene expression with exquisite temporal and spatial control and recent studies suggest that a large fraction of mRNAs localize to distinct cytoplasmic domains. In this review, we focus on cis-acting RNA localization elements, RNA-binding proteins, and the assembly of mRNAs into granules that are transported by molecular motors along cytoskeletal elements to their final destination in the cell. The process of mRNA localization and regulated translation has classically been considered to be a mechanism used by a handful of transcripts to spatially and temporally restrict gene expression to discrete sites within highly polarized, asymmetric cells. To date, the best-studied examples of mRNA localization all involve transcripts whose protein products play specialized roles within well-defined subcellular compartments. These include the mRNA encoding the transcriptional repressor ASH1 in budding yeast, which inhibits mating type switching. ASH1 mRNA is transported to the bud tip of a dividing cell such that it is delivered only to the nucleus of the daughter cell, thereby ensuring that the mother and daughter cells have distinct mating types (Paquin and Chartrand, 2008). In fruit fly Drosophila, the localization of mRNAs, such as bicoid, oskar, and nanos to anterior and posterior poles of the oocyte, helps establish morphogen gradients that underlie the proper spatial patterning of the developing embryo (Johnstone and Lasko, 2001). Similar processes occur in oocytes of the frog Xenopus, where localization of the mRNA encoding the T-box transcription factor VegT localizes to the vegetal pole and induces endodermal and mesodermal cell fates in the embryo (King et al., 2005). In fibroblasts, β-actin mRNA localizes to the lamellipodia, where its translation is required for cytoskeletal-mediated motility(Condeelis and Singer, 2005). In oligodendrocytes, the mRNA encoding myelin basic protein (MBP) is transported into the distal processes where myelination occurs (Smith, 2004). During brain development, local translation of mRNAs in axonal growth cones allows neurons to respond to local environmental cues as the distal axonal processes navigate towards their synaptic partners (Lin and Holt, 2007). In the mature brain, the regulated translation of synaptically localized mRNAs allows each of the thousands of synapses made by a given neuron to autonomously alter its structure and function during synaptic plasticity, thereby greatly enhancing the computational capacity of the brain (Martin and Zukin, 2006). Although these examples are of RNAs encoding proteins with specialized local functions, more recent studies indicate that the localization of mRNAs to particular subcellular compartments may be much more prevalent than previously thought. In a recent study involving highthroughput, high resolution in situ hybridizations of over 3,000 transcripts in Drosophila embryos, 71% were found to be expressed in spatially distinct patterns (Lecuyer et al., 2007).