RNA localization and co‐translational interactions control RAB 13 GTP ase function and cell migration
2020; Springer Nature; Volume: 39; Issue: 21 Linguagem: Inglês
10.15252/embj.2020104958
ISSN1460-2075
AutoresKonstadinos Moissoglu, Michael Stueland, Alexander N. Gasparski, Tianhong Wang, Lisa M. Jenkins, Michelle L. Hastings, Stavroula Mili,
Tópico(s)RNA Research and Splicing
ResumoArticle18 September 2020Open Access Source DataTransparent process RNA localization and co-translational interactions control RAB13 GTPase function and cell migration Konstadinos Moissoglu Konstadinos Moissoglu Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Michael Stueland Michael Stueland Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Alexander N Gasparski Alexander N Gasparski orcid.org/0000-0002-6012-4887 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Tianhong Wang Tianhong Wang Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Lisa M Jenkins Lisa M Jenkins Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Michelle L Hastings Michelle L Hastings orcid.org/0000-0002-4253-9261 Center for Genetic Diseases, Chicago Medical School, Rosalind Franklin University of Science and Medicine, North Chicago, IL, USA Search for more papers by this author Stavroula Mili Corresponding Author Stavroula Mili [email protected] orcid.org/0000-0002-9161-8660 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Konstadinos Moissoglu Konstadinos Moissoglu Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Michael Stueland Michael Stueland Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Alexander N Gasparski Alexander N Gasparski orcid.org/0000-0002-6012-4887 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Tianhong Wang Tianhong Wang Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Lisa M Jenkins Lisa M Jenkins Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Michelle L Hastings Michelle L Hastings orcid.org/0000-0002-4253-9261 Center for Genetic Diseases, Chicago Medical School, Rosalind Franklin University of Science and Medicine, North Chicago, IL, USA Search for more papers by this author Stavroula Mili Corresponding Author Stavroula Mili [email protected] orcid.org/0000-0002-9161-8660 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Author Information Konstadinos Moissoglu1, Michael Stueland1, Alexander N Gasparski1, Tianhong Wang1, Lisa M Jenkins2, Michelle L Hastings3 and Stavroula Mili *,1 1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA 2Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA 3Center for Genetic Diseases, Chicago Medical School, Rosalind Franklin University of Science and Medicine, North Chicago, IL, USA ‡This article has been contributed to by US Government employees and their work is in the public domain in the USA *Corresponding author. Tel: +1 240 760 6844; E-mail: [email protected] The EMBO Journal (2020)39:e104958https://doi.org/10.15252/embj.2020104958 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis The functional and mechanistic consequences of RNA localization to diverse subcellular compartments are unclear. Localization of mammalian RAB13 RNA at the cell periphery is required for RAB13 activation and mesenchymal cell migration, through co-translational interaction with the exchange factor RABIF. Peripherally-translated RAB13 has enhanced activity and promotes cell migration. Mislocalization of RAB13 RNA phenocopies complete loss of RAB13 protein in cell migration. Peripheral localization of RAB13 RNA promotes local co-translational interaction with the exchange factor RABIF. Introduction Localization of RNAs to diverse subcellular destinations is widely observed in various cell types and organisms (Meignin & Davis, 2010; Medioni et al, 2012; Buxbaum et al, 2015). However, in mammalian cells, the functional and mechanistic consequences are relatively unknown. In some cases, RNA accumulation can be accompanied by a corresponding increase in protein concentration at the same location. Such local protein gradients can be reinforced through translationally silencing RNAs prior to arrival at their destination (Besse & Ephrussi, 2008), thus ensuring tight spatial and temporal control of protein production and preventing deleterious effects of premature or ectopic translation (Jung et al, 2014; Buxbaum et al, 2015). This type of regulation has been described in highly polarized cells, such as neurons. For example, translational activation of RNAs localized at growth cones and the consequent increase in local protein abundance underlie axonal pathfinding decisions (Leung et al, 2006b; Colak et al, 2013; Wong et al, 2017). Similarly, activation of dendritic synapses upregulates translation of localized transcripts and is important for synaptic plasticity (Yoon et al, 2016; Rangaraju et al, 2017; Holt et al, 2019). Indeed, RNA localization appears to direct enrichment in neurites of almost half of the neurite-enriched proteome (Zappulo et al, 2017). A similar significant correlation between steady-state RNA and protein localization has been described in epithelial cells for proteins associated with organelles, such as mitochondria and the endoplasmic reticulum (Fazal et al, 2019). Nevertheless, a concordance between RNA localization and protein distribution is not always observed. One case in point concerns RNAs enriched at dynamic protrusions of mesenchymal-migrating cells. RNA localization at protrusions is important for protrusion stability and cell migration (Mili et al, 2008; Mardakheh et al, 2015; Wang et al, 2017). However, there is little correlation between RNA and protein distributions (Mardakheh et al, 2015) and protrusion-enriched RNAs can be similarly translated in both internal and peripheral locations (Moissoglu et al, 2019), thus raising the question of what the functional role of RNA transport in these cases is. Here, we investigate the consequences of local peripheral translation focusing on the RAB13 RNA. RAB13 is a member of the Rab family of small GTPases which play important roles in vesicle-mediated membrane trafficking (Ioannou & McPherson, 2016; Pfeffer, 2017). It is amplified in the majority of cancers, and its levels inversely correlate with prognosis (Ioannou & McPherson, 2016). Activation of RAB13 at the plasma membrane is required for cell migration and invasion (Ioannou et al, 2015), potentially through multiple mechanisms, including activity-dependent recycling of integrins or modulation of actin-binding proteins at the leading edge (Sakane et al, 2012, 2013; Sahgal et al, 2019). RAB13 RNA is prominently localized at protrusive regions of multiple cell types (Mili et al, 2008; Feltrin et al, 2012; Moissoglu et al, 2019) together with a group of RNAs whose localization is regulated by the adenomatous polyposis coli (APC) protein and detyrosinated microtubules (Wang et al, 2017). We have previously examined the translational regulation of the RAB13 RNA, showing that it is similarly translated in both internal and peripheral locations. Interestingly, translation of the RAB13 RNA at the periphery is dynamically regulated with the RNA being actively translated at extending protrusions, while undergoing silencing at retracting regions. Thus, peripheral RAB13 translation appears to be functionally linked with protrusive activity (Moissoglu et al, 2019). We show here that RAB13 RNA and protein distributions are quite discordant, with RAB13 RNA being enriched in the periphery, while RAB13 protein assumes mostly a perinuclear distribution. To assess the functional role of peripheral RNA localization, we devise a way to specifically prevent localization of RAB13 RNA at peripheral protrusions without affecting its translation, stability, or the localization of other co-regulated RNAs. Importantly, we show that peripheral RAB13 translation does not affect the overall distribution of the protein or its ability to associate with membranes but is required for activation of the GTPase and for efficient cell migration. Our data show that RAB13 associates co-translationally with the exchange factor RABIF. Peripheral translation is required for RABIF-RAB13 interaction at the periphery and for directing RAB13 GTPase activity to promote cell migration. Our results indicate that translation of RAB13 in specific subcellular environments imparts the protein with distinct properties, thus highlighting a means of controlling protein function through local RNA translation. Results RAB13 RNA and protein exhibit distinct subcellular distributions In both mouse and human mesenchymal cells, RAB13 RNA is prominently enriched at peripheral protrusions (Fig 1A, and Mili et al, 2008; Wang et al, 2017). Our prior work has shown that peripheral RAB13 RNA is actively translated at extending protrusions and silenced at retracting tails (Moissoglu et al, 2019). To assess whether translation of peripheral RAB13 RNA leads to a corresponding increase in RAB13 protein, we visualized the distribution of endogenous RAB13. Interestingly, despite the peripheral RAB13 RNA enrichment, at steady state, RAB13 protein is prominently concentrated around the nucleus (Fig 1B and C). However, since these cells are randomly migrating, some peripheral regions are in the process of retracting, thus likely containing silent RAB13 RNA (Moissoglu et al, 2019). To enrich for actively extending protrusions, we grew cells on microporous filters, induced them briefly to migrate toward the bottom surface, and assessed protein and RNA distributions between fractionated protrusions (Ps) and cell bodies (CB) (Fig 1D). Consistent with the imaging data above, RAB13 RNA is significantly enriched at extending protrusions while, still, RAB13 protein is not (Fig 1D). We additionally considered whether acute stimulation might lead to a transient increase in peripheral RAB13 protein, since RNA translation can be locally induced upon activation of specific cell surface receptors (Huttelmaier et al, 2005; Cagnetta et al, 2018; Koppers et al, 2019). Again, however, we do not observe any increase in the amount of RAB13 protein at protrusions or in the overall RAB13 protein levels, upon stimulation with serum (Fig EV1). Moreover, the reported half-life of RAB13 protein is several hours (Schwanhausser et al, 2011; Boisvert et al, 2012; Mathieson et al, 2018). It exists in the cytosol, in a pool that is expected to diffuse rapidly, and also associates with intracellular vesicles playing a role in vesicle trafficking. Therefore, we think it is reasonable to assume that most of the lifetime of a RAB13 protein would be spent away from its site of synthesis. Overall, while we cannot exclude the presence of an undetectable pool of peripheral protein with distinct regulation, we think that these results strongly suggest that at least a significant proportion of the protein translated from peripheral RAB13 RNA does not persist at the periphery but assumes a steady-state perinuclear distribution. Figure 1. RAB13 RNA and protein exhibit distinct subcellular distributions Representative FISH images showing RAB13 RNA distribution in MDA-MB-231 cells. Nuclei and cell outlines are shown in blue and green, respectively. Arrows point to RAB13 RNA concentrated at protrusive regions. Boxed regions are magnified in the insets. Representative immunofluorescence images of RAB13 protein in cells transfected with the indicated siRNAs. Reduction of intensity in RAB13 knockdown cells confirms the specificity of the signal. Arrows point to perinuclear RAB13 protein. Calibration bar shows intensity values. Ratios of peripheral/perinuclear intensity calculated from images as shown in (A) and (B). Bars: mean ± s.e.m. Values within each bar represent number of cells observed in 3 independent experiments. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate toward LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m. The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction. Data information: P-values: **< 0.01; ****< 0.0001 by Student's t-test (C) or analysis of variance with Dunnett's multiple comparisons test against GFP (D). Scale bars: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. RAB13 protein levels do not change upon serum stimulationMDA-MB-231 cells were stimulated with serum for the indicated times, in the presence or absence of cycloheximide (CHX). The cells were also treated with control or RAB13-mislocalizing PMOs. Representative Western blot analysis of whole-cell lysates and corresponding quantitations of RAB13 levels from n = 4–5 replicates. Bars: mean ± s.e.m. No significant differences by Friedman's test. Increase in pY397-FAK levels attests to serum stimulation. RAB13 immunofluorescence at representative protrusive regions. A cell line expressing GFP was used to delineate cell borders and provide an internal cytosolic control. RAB13 signal at front lamellipodial regions was quantified. n = 35–51 protrusions. Bars: mean ± s.e.m. No increase is detected upon stimulation. By contrast, at early time points a decrease is detected (5 and 20 min, P < 0.01 by Kruskal–Wallis test), potentially arising from serum-induced endocytosis of RAB13-containing membranes. Scale bars: 8 μm. Download figure Download PowerPoint A GA-rich motif within the mouse Rab13 3′UTR is necessary for localization at protrusions To understand the functional role of peripheral translation, we first sought to narrow down on specific localization sequences. We had previously shown that a 200–300-nt region of the mouse Rab13 3′UTR is sufficient for localization and can competitively inhibit the localization of other peripheral, APC-dependent RNAs (Wang et al, 2017), suggesting that it contains a binding site for a factor commonly bound to APC-dependent RNAs. Using sequence alignment and gazing, we noticed a particular GA-rich motif, with the consensus RGAAGRR (where R is a purine), which is present, in one or multiple copies, in the 3′UTR of the majority (~ 60%) of APC-dependent RNAs (Figs 2A and EV2) and which is significantly enriched [P = 4.99e-5; motif enrichment analysis (meme-suite.org)] in APC-dependent RNAs compared to APC-independent RNAs, an RNA group which is also enriched at protrusions but through a distinct pathway (Wang et al, 2017). To test for any functional significance, we expressed an exogenous RNA carrying either the wild-type Rab13 3′UTR or the 3′UTR carrying specific deletions of this motif (Fig 2A and B). We imaged RNAs using single-molecule FISH and measured a Peripheral Distribution Index (PDI) to quantify their distributions in multiple cells (Stueland et al, 2019). Consistent with previous observations (Wang et al, 2017), a control β-globin RNA shows a mostly diffuse cytoplasmic distribution (Fig 2B and C), while addition of the Rab13 3′UTR is sufficient to promote its peripheral localization, denoted by low and high PDI values, respectively. Interestingly, deletion of one RGAAGRR motif (Rab13 UTR (Δ1)) significantly perturbed the ability of the Rab13 3′UTR to direct localization of the β-globin RNA, while deletion of two of them (Rab13 UTR (Δ1 + 2)) had a stronger effect making the distribution of the reporter more similar to that of the non-localized control (Fig 2C). An endogenous localized RNA (Ddr2) remained similarly localized at protrusions in all conditions (Fig 2C). Therefore, at least some of the RGAAGRR motifs are required for peripheral localization. Figure 2. A GA-rich motif in the mouse Rab13 3′UTR is necessary for localization at protrusions Schematic showing the %GA content along the mouse Rab13 3′UTR using a 30-nt window size. Occurrences of the consensus GA-rich motif are indicated by a red rectangle. The exact sequence between nucleotides 153–216 is shown with GA motifs in red and deleted regions indicated by black bars. P-value by Fisher's exact test with Bonferroni's correction. FISH images of mouse fibroblasts expressing the β-globin coding sequence followed by the indicated UTRs. β-globin RNA is shown in yellow. Nuclei and cell outlines are shown in blue. Arrows point to β-globin RNA concentrated at protrusive regions. Δ1 and Δ1 + 2 indicate deletions of the regions shown in (A). Scale bars: 10 μm. Distribution of β-globin RNA or of Ddr2 RNA detected in the same cells, quantified by measuring a Peripheral Distribution Index (PDI). N = 35–55 cells observed in 3 independent experiments. Bars: mean ± 95% CI. ****P < 0.0001 by analysis of variance with Dunnett's multiple comparisons test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. GA-rich motif distribution in 3′UTRs of APC-dependent RNAsGraphs show the % GA content along the 3′UTR of the indicated APC-dependent RNAs using a 30-nt window size. The graph showing the Rab13 UTR is the same presented in Figs 2A and 3A. Occurrences of the consensus GA-rich motif are indicated by a red rectangle. Wider rectangles indicate the presence of multiple motifs. Exact sizes are not to scale due to the variable UTR lengths. The majority of GA-rich motifs are found within more extended GA-rich regions with GA content > 75%. Note that the GA motif is 7 nts, while the window for %GA calculation is 30 nts. Download figure Download PowerPoint Antisense oligonucleotides against the GA-rich region specifically interfere with localization of Rab13 RNA Another notable feature of these motifs is that the majority of them (62%; 148 of 239 motifs in 3′UTRs of mouse APC-dependent RNAs) are found within more extended GA-rich regions, which exhibit high GA content (> 75%) for at least 30 consecutive nucleotides or more (Fig EV2). To further investigate the roles of these different features and to, at the same time, interfere with the localization of the endogenous Rab13 RNA, we used antisense oligonucleotides (ASOs), which can interfere with RNA structure formation or RNA–protein binding (Hua et al, 2010; Lentz et al, 2013; Havens & Hastings, 2016) (Fig 3A). Here, we utilized 25-nt-long phosphorodiamidate morpholino (PMO) ASOs. Figure 3. Antisense oligonucleotides against the GA-rich region specifically interfere with localization of Rab13 RNA Schematic showing positions along the mouse Rab13 3′UTR targeted by the indicated PMOs. PMOs #1, 2, and 3 target the RGAAGRR motifs or the adjacent GA-rich region. PMOs #6 and #7 target the Rab13 3′UTR outside of the GA-rich region. The control PMO targets an intronic sequence of human β-globin. Red rectangles and text indicate the location of the GA-rich motifs. FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs. Cyb5r3 is an APC-dependent RNA also enriched at protrusions. Arrows: peripheral Rab13 RNA. Arrowheads: perinuclear Rab13 RNA. Boxed regions are magnified in the insets. Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region. Scale bars: 10 μm. (4 μm in insets). ****P < 0.0001 by analysis of variance with Dunnett's multiple comparisons test. N = 40–90 cells observed in 3–6 independent experiments. Bars: mean ± 95% CI. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n = 3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **P = 0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells (n = 4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls. Download figure Download PowerPoint We first delivered fluorescently labeled PMOs to determine the efficiency of delivery and their persistence in cells. PMOs were taken up by virtually all cells and persisted, either within apparent endosomal structures or released into the cytosol, for more than 3 days (Appendix Fig S1A and B). The effect of antisense PMOs on the localization of Rab13 RNA was assessed, 3 days after PMO delivery, by single-molecule FISH of the endogenous Rab13 RNA and PDI calculation. As expected, cells exposed to the control PMO exhibited peripheral localization of Rab13 RNA. Similarly, the Rab13 #6 and #7 PMOs did not affect Rab13 RNA distribution (Fig 3B). However, PMOs targeting the RGAAGRR motifs (PMOs #2 and #3) caused a pronounced mislocalization of Rab13 RNA toward the perinuclear cytoplasm, evidenced by a significant reduction in PDI values (Fig 3B). Interestingly, the Rab13 PMO #1, which targets the adjacent GA-rich region, disrupted localization to a similar extent, suggesting that apart from the RGAAGRR motifs additional GA-rich sequences are important for localization or that the overall structure of this region is important. Notably, within the same cells, another APC-dependent RNA, Cyb5r3, which also contains GA-rich motifs, maintained its localization at protrusions under all conditions. Therefore, PMOs against the GA-rich region of Rab13 RNA appear to specifically perturb Rab13 RNA localization at protrusions. To more extensively investigate the specificity of the observed effect, we assessed the distribution of a panel of ~ 20 APC-dependent RNAs, as well as of several APC-independent RNAs, using a protrusion/cell body fractionation scheme (Wang et al, 2017). As described previously (Wang et al, 2017), APC-dependent RNAs are enriched at protrusions, and their enrichment is more pronounced than that exhibited by APC-independent RNAs (Fig 3C). Importantly, cells treated with a mislocalizing Rab13 PMO exhibited indistinguishable distributions for all RNAs tested, with the notable exception of the targeted Rab13 RNA, which became significantly less enriched at protrusions, corroborating and extending the FISH analysis described above (Fig 3B). We conclude that antisense PMOs against the Rab13 GA-rich region specifically alter the distribution of Rab13 RNA without impacting the distribution of other RNAs, even those belonging to the same co-regulated group. We additionally examined the overall abundance of the same panel of RNAs. PMO oligos do not trigger RNase H activity, and consistent with that, we did not observe any detectable change in the total levels of either Rab13 or any other RNA, in cells treated with Rab13 PMOs (Fig 3D and Appendix Fig S2). Therefore, this approach allows us to specifically alter the distribution of the endogenous Rab13 RNA without affecting its overall abundance in cells. The human RAB13 3′UTR exhibits a functionally conserved GA-rich region required for peripheral localization RAB13 RNA is localized at protrusive regions in diverse cell types and species. We thus sought whether similar sequence determinants support localization of the human RAB13 transcript, which exhibits a GA-rich region (> 75%) and interspersed RGAAGRR motifs with similar topology as that of the mouse Rab13 3′UTR sequence (nts 98–268; Fig 4A). To identify functional regions with regard to RNA localization at protrusions, we delivered PMOs targeting regions across the length of the 3′UTR. PMOs targeting either RGAAGRR motifs directly (RAB13 PMOs 165 and 230) or adjacent GA-rich regions (RAB13 PMOs 91, 113, 191, and 210) (Fig 4A) significantly affected RAB13 RNA localization. By contrast, all PMOs targeting sites outside of the GA-rich region did not affect RNA distribution (Fig 4A). Concomitant delivery of two individual PMOs (RAB13 PMOs 191 and 230) had an additive effect resulting in marked RAB13 RNA mislocalization. Furthermore, the observed effects were specific for RAB13 RNA since the distribution of another peripherally localized RNA, NET1, was not affected (Fig 4A and B). Thus, also in human cells, interfering with either the RGAAGRR motifs or the adjacent GA-rich regions specifically perturbs the peripheral localization of RAB13 RNA. Figure 4. The human RAB13 3′UTR contains a functionally conserved GA-rich region required for peripheral localization Schematic showing %GA content and positions along the human RAB13 3′UTR targeted by the indicated PMOs. Red rectangles indicate the location of GA-rich motifs. Graphs present PDI measurements of RAB13 RNA (upper panel) or NET1 RNA (another APC-dependent RNA; bottom panel) detected in MDA-MB-231 cells treated with the indicated PMOs. PDI = 1 indicates a diffuse distribution. P-values: **< 0.01, ***< 0.001, ****< 0.0001 by analysis of variance with Dunnett's multiple comparisons test. n.s.: non-significant. N = 30–73 cells observed in 3–5 independent experiments. Bars: mean ± s.e.m. Representative FISH images of cells treated with the indicated PMOs. Arrows point to peripheral RNA. Arrowheads point to perinuclear RNA. Boxed regions are magnified in the insets. Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region, while NET1 remains localized at protrusions. Scale bars: 10 μm (4 μm in insets). RAB13 protein levels were measured by quantitative Western blot and normalized to total α-tubulin or GAPDH levels (for representative blot, see Fig 5E). Relative levels in RAB13 PMO-treated cells compared to control are shown. No significant differences were detected by Kruskal–Wallis test with Dunn's multiple comparisons test. N = 3–8. Bars: mean ± SD. Download figure Download PowerPoint Importantly, RNA mislocalization was not accompanied by any detectable change in the amount of RAB13 protein produced (Fig 4C). Specifically, the same amount of RAB13 protein is produced under basal conditions in cells exhibiting either peripheral (control PMO) or perinuclear (RAB13 PMO 230 or 191 + 230) RAB13 RNA distribution (Fig 4C). This is consistent with the recently reported observation that RAB13 RNA is similarly translated in both perinuclear and peripheral regions (Moissoglu et al, 2019). Furthermore, the location of the RAB13 RNA did not affect the total RAB13 protein levels or the amount of RAB13 protein at protrusions, upon acute stimulation with serum (Fig EV1). We conclude that, under both basal and stimulated conditions, the use of ASOs allows us to specifically assess the functional roles promoted by the localization of RAB13 RNA without confounding contributions due to altered protein expression. Peripheral RAB13 RNA localization is important for cell migration To understand the functional role of RAB13 RNA localization at protrusions, we assessed the effect of RAB13 RNA mislocalization on the ability of cells to migrate, given that the encoded RAB13 protein promotes cell migration through multiple mechanisms (Ioannou & McPherson, 2016). For this, we compared cells treated with control PMOs or RAB13 mislocalizing PMOs using various assays. In one case, cells plated on microporous Transwell membrane inserts were induced to migrate toward a chemoattractant gradient and the numbe
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