mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

RNA localization and co-translational interactions control RAB13 GTPase function and cell migration.

Numerous RNAs exhibit specific distribution patterns in mammalian cells. However, the functional and mechanistic consequences are relatively unknown. Here, we investigate the functional role of RNA localization at cellular protrusions of migrating mesenchymal cells, using as a model the RAB13 RNA, which encodes a GTPase important for vesicle-mediated membrane trafficking. While RAB13 RNA is enriched at peripheral protrusions, the expressed protein is concentrated perinuclearly. By specifically preventing RAB13 RNA localization, we show that peripheral RAB13 translation is not important for the overall distribution of the RAB13 protein or its ability to associate with membranes, but is required for full activation of the GTPase and for efficient cell migration. RAB13 translation leads to a co-translational association of nascent RAB13 with the exchange factor RABIF. Our results indicate that RAB13-RABIF association at the periphery is required for directing RAB13 GTPase activity to promote cell migration. Thus, translation of RAB13 in specific subcellular environments imparts the protein with distinct properties and highlights a means of controlling protein function through local RNA translation.

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.

Polarity and differential inheritance--universal attributes of life?

When and why did cell polarization arise? Recent work in bacteria and yeast suggests that polarization may have evolved to restrict senescence to one daughter during division by enabling the differential segregation of damaged material. In more complex organisms, polarity functions have diversified to permit the differential inheritance of centrosomes, RNAs, proteins, and membranes, which is essential for the generation of diverse cell types from stem cells and for morphogenesis.

Collective cancer cell invasion requires RNA accumulation at the invasive front.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions.

RNA localization is important for the establishment and maintenance of polarity in multiple cell types. Localized RNAs are usually transported along microtubules or actin filaments and become anchored at their destination to some underlying subcellular structure. Retention commonly involves actin or actin-associated proteins, although cytokeratin filaments and dynein anchor certain RNAs. RNA localization is important for diverse processes ranging from cell fate determination to synaptic plasticity; however, so far there have been few comprehensive studies of localized RNAs in mammalian cells. Here we have addressed this issue, focusing on migrating fibroblasts that polarize to form a leading edge and a tail in a process that involves asymmetric distribution of RNAs. We used a fractionation scheme combined with microarrays to identify, on a genome-wide scale, RNAs that localize in protruding pseudopodia of mouse fibroblasts in response to migratory stimuli. We find that a diverse group of RNAs accumulates in such pseudopodial protrusions. Through their 3' untranslated regions these transcripts are anchored in granules concentrated at the plus ends of detyrosinated microtubules. RNAs in the granules associate with the adenomatous polyposis coli (APC) tumour suppressor and the fragile X mental retardation protein (FMRP). APC is required for the accumulation of transcripts in protrusions. Our results suggest a new type of RNA anchoring mechanism as well as a new, unanticipated function for APC in localizing RNAs.

A KIF1C-CNBP motor-adaptor complex for trafficking mRNAs to cell protrusions.

mRNA localization to subcellular compartments is a widely used mechanism that functionally contributes to numerous processes. mRNA targeting can be achieved upon recognition of RNA cargo by molecular motors. However, our molecular understanding of how this is accomplished is limited, especially in higher organisms. We focus on a pathway that targets mRNAs to peripheral protrusions of mammalian cells and is important for cell migration. Trafficking occurs through active transport on microtubules, mediated by the KIF1C kinesin. Here, we identify the RNA-binding protein CNBP, as a factor required for mRNA localization to protrusions. CNBP binds directly to GA-rich sequences in the 3'UTR of protrusion targeted mRNAs. CNBP also interacts with KIF1C and is required for KIF1C recruitment to mRNAs and for their trafficking on microtubules to the periphery. This work provides a molecular mechanism for KIF1C recruitment to mRNA cargo and reveals a motor-adaptor complex for mRNA transport to cell protrusions.

Visualizing and Quantifying mRNA Localization at the Invasive Front of 3D Cancer Spheroids.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

RNA localization in confined cells depends on cellular mechanical activity and contributes to confined migration.

Cancer cells experience mechanical confining forces during metastasis and, consequently, can alter their migratory mechanisms. Localization of numerous mRNAs to cell protrusions contributes to cell polarization and migration and is controlled by proteins that can bind RNA and/or cytoskeletal elements, such as the adenomatous polyposis coli (APC). Here, we demonstrate that peripheral localization of APC-dependent RNAs in cells within confined microchannels is cell type dependent. This varying phenotype is determined by the levels of a detyrosinated tubulin network. We show that this network is regulated by mechanoactivity and that cells with mechanosensitive ion channels and increased myosin II activity direct peripheral localization of the RAB13 APC-dependent RNA. Through specific mislocalization of the RAB13 RNA, we show that peripheral RNA localization contributes to confined cell migration. Our results indicate that a cell's mechanical activity determines its ability to peripherally target RNAs and utilize them for movement in confinement.

Regulation and outcomes of localized RNA translation.

Spatial segregation of mRNAs in the cytoplasm of cells is a well-known biological phenomenon that is widely observed in diverse species spanning different kingdoms of life. In mammalian cells, localization of mRNAs has been documented and studied quite extensively in highly polarized cells, most notably in neurons, where localized mRNAs function to direct protein production at sites that are quite distant from the soma. Recent studies have strikingly revealed that a large proportion of the cellular transcriptome exhibits polarized distributions even in cells that lack an obvious need for long-range transport, such as fibroblasts or epithelial cells. This review focuses on emerging concepts regarding the functional outcomes of mRNA targeting in the cytoplasm of such cells. We also discuss regulatory mechanisms controlling these events, with an emphasis on the role of cell mechanics and the organization of the cytoskeleton. This article is categorized under: Translation > Regulation RNA Export and Localization > RNA Localization.

EGF receptor-mediated FUS phosphorylation promotes its nuclear translocation and fibrotic signaling.

Excessive accumulation of collagen leads to fibrosis. Integrin α1ß1 (Itgα1ß1) prevents kidney fibrosis by reducing collagen production through inhibition of the EGF receptor (EGFR) that phosphorylates cytoplasmic and nuclear proteins. To elucidate how the Itgα1ß1/EGFR axis controls collagen synthesis, we analyzed the levels of nuclear tyrosine phosphorylated proteins in WT and Itgα1-null kidney cells. We show that the phosphorylation of the RNA-DNA binding protein fused in sarcoma (FUS) is higher in Itgα1-null cells. FUS contains EGFR-targeted phosphorylation sites and, in Itgα1-null cells, activated EGFR promotes FUS phosphorylation and nuclear translocation. Nuclear FUS binds to the collagen IV promoter, commencing gene transcription that is reduced by inhibiting EGFR, down-regulating FUS, or expressing FUS mutated in the EGFR-targeted phosphorylation sites. Finally, a cell-penetrating peptide that inhibits FUS nuclear translocation reduces FUS nuclear content and collagen IV transcription. Thus, EGFR-mediated FUS phosphorylation regulates FUS nuclear translocation and transcription of a major profibrotic collagen gene. Targeting FUS nuclear translocation offers a new antifibrotic therapy.

RDI Calculator: An Analysis Tool to Assess RNA Distributions in Cells.

Localization of RNAs to various subcellular destinations has emerged as a widely used mechanism that regulates a large proportion of transcripts in polarized cells. A number of methodologies have been developed that allow detection and imaging of RNAs at single-molecule resolution. However, methodologies to quantitatively describe RNA distributions are limited. Such approaches usually rely on the identification of cytoplasmic and nuclear boundaries which are used as reference points. Here, we describe an automated, interactive image analysis program that facilitates the accurate generation of cellular outlines from single cells and the subsequent calculation of metrics that quantify how a population of RNA molecules is distributed in the cell cytoplasm. We apply this analysis to mRNAs in mouse and human cells to demonstrate how these metrics can highlight differences in the distribution patterns of distinct RNA species. We further discuss considerations for the practical use of this tool. This program provides a way to facilitate and expedite the analysis of subcellular RNA localization for mechanistic and functional studies.

Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport.

Localization of RNAs to various subcellular destinations is a widely used mechanism that regulates a large proportion of transcripts in polarized cells. In many cases, such localized transcripts mediate spatial control of gene expression by being translationally silent while in transit and locally activated at their destination. Here, we investigate the translation of RNAs localized at dynamic cellular protrusions of human and mouse, migrating, mesenchymal cells. In contrast to the model described above, we find that protrusion-localized RNAs are not locally activated solely at protrusions, but can be translated with similar efficiency in both internal and peripheral locations. Interestingly, protrusion-localized RNAs are translated at extending protrusions, they become translationally silenced in retracting protrusions and this silencing is accompanied by coalescence of single RNAs into larger heterogeneous RNA clusters. This work describes a distinct mode of translational regulation of localized RNAs, which we propose is used to regulate protein activities during dynamic cellular responses.

LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain, is bound in vivo to mitochondrial and nuclear RNAs.

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.

Extracellular matrix stiffness and cell contractility control RNA localization to promote cell migration.

Numerous RNAs are enriched within cellular protrusions, but the underlying mechanisms are largely unknown. We had shown that the APC (adenomatous polyposis coli) protein controls localization of some RNAs at protrusions. Here, using protrusion-isolation schemes and RNA-Seq, we find that RNAs localized in protrusions of migrating fibroblasts can be distinguished in two groups, which are differentially enriched in distinct types of protrusions, and are additionally differentially dependent on APC. APC-dependent RNAs become enriched in high-contractility protrusions and, accordingly, their localization is promoted by increasing stiffness of the extracellular matrix. Dissecting the underlying mechanism, we show that actomyosin contractility activates a RhoA-mDia1 signaling pathway that leads to formation of a detyrosinated-microtubule network, which in turn is required for localization of APC-dependent RNAs. Importantly, a competition-based approach to specifically mislocalize APC-dependent RNAs suggests that localization of the APC-dependent RNA subgroup is functionally important for cell migration.Adenomatous polyposis coli (APC) regulates the localization of some mRNAs at cellular protrusions but the underlying mechanisms and functional roles are not known. Here the authors show that APC-dependent RNAs are enriched in contractile protrusions, via detyrosinated microtubules, and enhance cell migration.

FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination.

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)-linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)-microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

Dysregulated axonal RNA translation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease that has been associated with a diverse array of genetic changes. Prominent among these are mutations in RNA-binding proteins (RBPs) or repeat expansions that give rise to toxic RNA species. RBPs are additionally central components of pathologic aggregates that constitute a disease hallmark, suggesting that dysregulation of RNA metabolism underlies disease progression. In the context of neuronal physiology, transport of RNAs and localized RNA translation in axons are fundamental to neuronal survival and function. Several lines of evidence suggest that axonal RNA translation is a central process perturbed by various pathogenic events associated with ALS. Dysregulated translation of specific RNA groups could underlie feedback effects that connect and reinforce disease manifestations. Among such candidates are RNAs encoding proteins involved in the regulation of microtubule dynamics. Further understanding of axonally dysregulated RNA targets and of the feedback mechanisms they induce could provide useful therapeutic insights. WIREs RNA 2016, 7:589-603. doi: 10.1002/wrna.1352 For further resources related to this article, please visit the WIREs website.

The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules.

RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. In one such pathway the tumor-suppressor protein adenomatous polyposis coli (APC) targets RNAs to cell protrusions, forming APC-containing ribonucleoprotein complexes (APC-RNPs). Here, we show that APC-RNPs associate with the RNA-binding protein Fus/TLS (fused in sarcoma/translocated in liposarcoma). Fus is not required for APC-RNP localization but is required for efficient translation of associated transcripts. Labeling of newly synthesized proteins revealed that Fus promotes translation preferentially within protrusions. Mutations in Fus cause amyotrophic lateral sclerosis (ALS) and the mutant protein forms inclusions that appear to correspond to stress granules. We show that overexpression or mutation of Fus results in formation of granules, which preferentially recruit APC-RNPs. Remarkably, these granules are not translationally silent. Instead, APC-RNP transcripts are translated within cytoplasmic Fus granules. These results unexpectedly show that translation can occur within stress-like granules. Importantly, they identify a new local function for cytoplasmic Fus with implications for ALS pathology.

RNA localization and polarity: from A(PC) to Z(BP).

Cell polarization relies on the asymmetric organization of cellular structures and activities, and is fundamentally important both during development and for the proper function of most somatic cells. Asymmetries in the distribution and activity of proteins can be achieved through localization of RNA molecules that usually give rise to proteins at specific subcellular sites. It is increasingly appreciated that this is a widely used mechanism affecting protein function at multiple levels. The description of a new RNA localization pathway involving the tumor-suppressor protein APC raises questions regarding coordination between distinct localization pathways and their effects on protein function and cell polarity.