G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

Cell Reports, Journal Year: 2025, Volume and Issue: 44(2), P. 115237 - 115237

Published: Feb. 1, 2025

The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, β-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) support localized synthesis, crucial for However, whether other RBPs also localize at FAs remains unclear. Here, we identify hundreds that enriched (FA-mRNAs). FA-mRNAs share characteristics with stress granule (SG) found ribonucleoprotein (RNP) complexes the SG RBP. Mechanistically, G3BP1 binds FA proteins an RNA-dependent manner, dimerization domains, essential form RNPs SG, required We find promote speed by enhancing mobility size. These findings suggest previously unappreciated regulating function under non-stress conditions.

Language: Английский

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Regulation and outcomes of localized RNA translation

Wiley Interdisciplinary Reviews - RNA, Journal Year: 2022, Volume and Issue: 13(6)

Published: Feb. 14, 2022

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

Language: Английский

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Single-cell transcriptional dynamics in a living vertebrate

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 5, 2024

The ability to quantify transcriptional dynamics in individual cells via live imaging has revolutionized our understanding of gene regulation. However, such measurements are lacking the context vertebrate embryos. We addressed this deficit by applying MS2-MCP mRNA labeling quantification transcription zebrafish, a model vertebrate. developed platform transgenic organisms, light sheet fluorescence microscopy, and optimized image analysis that enables visualization MS2 reporters. used these tools obtain first single-cell, real-time segmentation clock. Our challenge traditional view smooth clock oscillations instead suggest discrete bursts organized space time. Together, results highlight how measuring single-cell activity can reveal unexpected features regulation data fuel dialogue between theory experiment.

Language: Английский

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Mechanistic insights into the basis of widespread RNA localization

Nature Cell Biology, Journal Year: 2024, Volume and Issue: 26(7), P. 1037 - 1046

Published: July 1, 2024

Language: Английский

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The kinesin-3 KIF1C undergoes liquid-liquid phase separation for accumulation of specific transcripts at the cell periphery

The EMBO Journal, Journal Year: 2024, Volume and Issue: 43(15), P. 3192 - 3213

Published: June 19, 2024

Language: Английский

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RNA localization and co‐translational interactions control RAB 13 GTP ase function and cell migration

The EMBO Journal, Journal Year: 2020, Volume and Issue: 39(21)

Published: Sept. 18, 2020

Article18 September 2020Open Access Source DataTransparent process RNA localization and co-translational interactions control RAB13 GTPase function cell migration Konstadinos Moissoglu Laboratory of Cellular Molecular Biology, Center for Cancer Research, National Institute, NIH, Bethesda, MD, USA Search more papers by this author Michael Stueland Alexander N Gasparski orcid.org/0000-0002-6012-4887 Tianhong Wang Lisa M Jenkins Cell Michelle L Hastings orcid.org/0000-0002-4253-9261 Genetic Diseases, Chicago Medical School, Rosalind Franklin University Science Medicine, North Chicago, IL, Stavroula Mili Corresponding Author [email protected] orcid.org/0000-0002-9161-8660 Information Moissoglu1, Stueland1, Gasparski1, Wang1, Jenkins2, Hastings3 *,1 1Laboratory 2Laboratory 3Center ‡This article has been contributed to US Government employees their work is in the public domain *Corresponding author. Tel: +1 240 760 6844; E-mail: The EMBO Journal (2020)39:e104958https://doi.org/10.15252/embj.2020104958 PDFDownload PDF text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Numerous RNAs exhibit specific distribution patterns mammalian cells. However, functional mechanistic consequences are relatively unknown. Here, we investigate role at cellular protrusions migrating mesenchymal cells, using as model RNA, which encodes important vesicle-mediated membrane trafficking. While enriched peripheral protrusions, expressed protein concentrated perinuclearly. By specifically preventing localization, show that translation not overall or its ability associate with membranes, but required full activation efficient migration. leads association nascent exchange factor RABIF. Our results indicate RAB13-RABIF periphery directing activity promote Thus, subcellular environments imparts distinct properties highlights means controlling through local translation. Synopsis diverse compartments unclear. Localization migration, interaction Peripherally-translated enhanced promotes Mislocalization phenocopies complete loss Peripheral Introduction destinations widely observed various types organisms (Meignin Davis, 2010; Medioni et al, 2012; Buxbaum 2015). In some cases, accumulation can be accompanied corresponding increase concentration same location. Such gradients reinforced translationally silencing prior arrival destination (Besse Ephrussi, 2008), thus ensuring tight spatial temporal production deleterious effects premature ectopic (Jung 2014; This type regulation described highly polarized such neurons. For example, translational localized growth cones consequent abundance underlie axonal pathfinding decisions (Leung 2006b; Colak 2013; Wong 2017). Similarly, dendritic synapses upregulates transcripts synaptic plasticity (Yoon 2016; Rangaraju 2017; Holt 2019). Indeed, appears direct enrichment neurites almost half neurite-enriched proteome (Zappulo A similar significant correlation between steady-state epithelial cells proteins associated organelles, mitochondria endoplasmic reticulum (Fazal Nevertheless, concordance always observed. One case point concerns dynamic mesenchymal-migrating protrusion stability (Mili 2008; Mardakheh 2015; there little distributions (Mardakheh 2015) protrusion-enriched similarly translated both internal locations (Moissoglu 2019), raising question what transport these cases is. focusing on RNA. member Rab family small GTPases play roles trafficking (Ioannou McPherson, Pfeffer, It amplified majority cancers, levels inversely correlate prognosis 2016). Activation plasma invasion 2015), potentially multiple mechanisms, activity-dependent recycling integrins modulation actin-binding leading edge (Sakane 2012, Sahgal prominently protrusive regions Feltrin 2019) together group whose regulated adenomatous polyposis coli (APC) detyrosinated microtubules (Wang We have previously examined showing it locations. Interestingly, dynamically being actively extending while undergoing retracting regions. functionally linked here quite discordant, periphery, assumes mostly perinuclear distribution. To assess devise way prevent without affecting translation, stability, other co-regulated RNAs. Importantly, does affect membranes data associates co-translationally RABIF-RAB13 properties, highlighting Results mouse human (Fig 1A, shown silenced tails whether protein, visualized endogenous RAB13. despite enrichment, steady state, around nucleus 1B C). since randomly migrating, retracting, likely containing silent enrich grew microporous filters, induced them briefly migrate toward bottom surface, assessed fractionated (Ps) bodies (CB) 1D). Consistent imaging above, significantly while, still, additionally considered acute stimulation might lead transient locally upon surface receptors (Huttelmaier 2005; Cagnetta 2018; Koppers Again, however, do observe any amount levels, serum EV1). Moreover, reported half-life several hours (Schwanhausser 2011; Boisvert Mathieson 2018). exists cytosol, pool expected diffuse rapidly, also intracellular vesicles playing vesicle Therefore, think reasonable assume most lifetime would spent away from site synthesis. Overall, cannot exclude presence an undetectable regulation, strongly suggest least proportion persist Figure 1. Representative FISH images MDA-MB-231 Nuclei outlines blue green, respectively. Arrows Boxed magnified insets. immunofluorescence transfected indicated siRNAs. Reduction intensity knockdown confirms specificity signal. protein. Calibration bar shows values. Ratios peripheral/perinuclear calculated (A) (B). Bars: mean ± s.e.m. Values within each represent number 3 independent experiments. Protrusions LPA were isolated analyzed detect (by Western blot; left panels) RT-ddPCR; right panel). Ps/CB ratios 2 experiments shown. pY397-FAK serves verify newly formed adhesions Ps fraction. Data information: P-values: **< 0.01; ****< 0.0001 Student's t-test (C) analysis variance Dunnett's comparisons test against GFP (D). Scale bars: 10 μm. Download figure PowerPoint Click expand figure. EV1. change stimulationMDA-MB-231 stimulated times, absence cycloheximide (CHX). treated RAB13-mislocalizing PMOs. blot whole-cell lysates quantitations n = 4–5 replicates. No differences Friedman's test. Increase attests stimulation. representative line expressing was used delineate borders provide cytosolic control. signal front lamellipodial quantified. 35–51 protrusions. detected contrast, early time points decrease (5 20 min, P < 0.01 Kruskal–Wallis test), arising serum-induced endocytosis RAB13-containing membranes. 8 GA-rich motif Rab13 3′UTR necessary understand first sought narrow down sequences. had 200–300-nt region sufficient competitively inhibit peripheral, APC-dependent 2017), suggesting contains binding commonly bound Using sequence alignment gazing, noticed particular motif, consensus RGAAGRR (where R purine), present, one copies, (~ 60%) (Figs 2A EV2) [P 4.99e-5; (meme-suite.org)] compared APC-independent RNAs, pathway significance, exogenous carrying either wild-type deletions B). imaged single-molecule measured Distribution Index (PDI) quantify (Stueland previous observations β-globin cytoplasmic 2B C), addition denoted low high PDI values, deletion (Rab13 UTR (Δ1)) perturbed two (Δ1 + 2)) stronger effect making reporter non-localized 2C). An (Ddr2) remained all conditions motifs localization. 2. Schematic %GA content along 30-nt window size. Occurrences red rectangle. exact nucleotides 153–216 GA deleted black bars. P-value Fisher's Bonferroni's correction. fibroblasts coding followed UTRs. yellow. blue. Δ1 (A). Ddr2 quantified measuring (PDI). 35–55 95% CI. ****P EV2. 3′UTRs RNAsGraphs % graph presented Figs 3A. Wider rectangles motifs. Exact sizes scale due variable lengths. found extended > 75%. Note 7 nts, calculation 30 nts. Antisense oligonucleotides interfere Another notable feature (62%; 148 239 RNAs) regions, (> 75%) consecutive EV2). further different features to, time, antisense (ASOs), structure formation RNA–protein (Hua Lentz Havens Hastings, 2016) 3A). utilized 25-nt-long phosphorodiamidate morpholino (PMO) ASOs. 3. positions targeted PMOs #1, 2, target adjacent region. #6 #7 outside PMO targets intronic β-globin. Red location measurements fibroblast Cyb5r3 Arrows: Arrowheads: becomes (4 μm insets). 40–90 3–6 Protrusion body fractions Rab13-PMO #2. nanoString calculate (n 3; s.e.m.). only affected. **P two-way ANOVA Levels determined control- #2-treated 4; controls. delivered fluorescently labeled determine efficiency delivery persistence taken up virtually persisted, apparent endosomal structures released into than days (Appendix Fig S1A assessed, after delivery, calculation. As expected, exposed exhibited did 3B). targeting (PMOs #2 #3) caused pronounced mislocalization cytoplasm, evidenced reduction values region, disrupted extent, apart additional sequences important. Notably, another Cyb5r3, motifs, maintained under conditions. appear perturb extensively effect, panel ~ well protrusion/cell fractionation scheme 3C). mislocalizing indistinguishable tested, exception became less corroborating above conclude alter impacting even those belonging group. oligos trigger RNase H activity, consistent that, detectable total 3D Appendix S2). approach allows us exhibits conserved species. determinants support transcript, interspersed topology (nts 98–268; 4A). identify regard across length 3′UTR. directly (RAB13 165 230) 91, 113, 191, 210) 4A) affected sites Concomitant individual 191 additive resulting marked mislocalization. Furthermore, peripherally NET1, 4A interfering perturbs 4. Graphs present (upper panel) NET1 (another RNA; 1 indicates 0.01, ***< 0.001, n.s.: non-significant. 30–73 3–5 Arrowheads remains quantitative normalized α-tubulin GAPDH (for blot, see 5E). Relative PMO-treated Dunn's 3–8. SD. produced 4C). Specifically, basal exhibiting (control PMO) 230 recently observation conditions, use ASOs promoted confounding contributions altered expression. migrate, given encoded mechanisms this, assays. case, plated Transwell inserts chemoattractant gradient numbe

Language: Английский

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Collective cancer cell invasion requires RNA accumulation at the invasive front

Proceedings of the National Academy of Sciences, Journal Year: 2020, Volume and Issue: 117(44), P. 27423 - 27434

Published: Oct. 15, 2020

Significance Specific RNAs are enriched at protrusive regions of migrating cells. This localization is important for cell migration on 2D surfaces. However, in vivo, tumor cells navigate complex 3D environments often collective groups. Here, we investigated protrusion-enriched during invasion. We show that specific exhibit a striking accumulation the front invasive leader provide insights into mechanism underlying RNA front, and further demonstrate it required efficient invasion additionally observe enrichment sites vivo tumors, supporting physiological relevance this suggesting targeting opportunity perturbing cancer

Language: Английский

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The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

RNA, Journal Year: 2021, Volume and Issue: 27(12), P. 1528 - 1544

Published: Sept. 7, 2021

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 mechanism. We found that the KIF1C motor interacts is required their localization. Live imaging revealed rapid, active transport single over long distances requires both microtubules KIF1C. Two-color directly transported by motors, 3′UTR being sufficient trigger KIF1C-dependent Moreover, remained associated peripheral, multimeric clusters was formation. These results reveal widespread pathway mammalian cells, which has dual role transporting RNAs clustering them within protrusions. Interestingly, also transports its own mRNA, suggesting possible feedback loop acting at level mRNA transport.

Language: Английский

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Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: June 9, 2023

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes neurites through unknown mechanisms. We find that MBNL forms motile anchored granules neurons myoblasts, selectively associates with kinesins Kif1bα Kif1c its zinc finger (ZnF) domains. Other RBPs similar ZnFs associate these kinesins, implicating a motor-RBP specificity code. kinesin perturbation leads widespread mRNA mis-localization, including depletion Nucleolin transcripts from neurites. Live cell imaging fractionation reveal the unstructured carboxy-terminal tail MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment Imaging (RBP-MRI), reconstitutes kinesin- membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple association, binding, membrane while establishing general strategies studying multi-functional, modular domains RBPs.

Language: Английский

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Localization ofKif1cmRNA to cell protrusions dictates binding partner specificity of the encoded protein

Genes & Development, Journal Year: 2023, Volume and Issue: 37(5-6), P. 191 - 203

Published: March 1, 2023

Subcellular localization of messenger RNA (mRNA) is a widespread phenomenon that can impact the regulation and function encoded protein. In nonneuronal cells, specific mRNAs localize to cell protrusions, proper mRNA required for migration. However, mechanisms by which regulates protein in this setting remain unclear. Here, we examined functional consequences encoding KIF1C. KIF1C kinesin motor migration trafficking, including trafficking its own mRNA. We show Kif1c does not regulate KIF1C's abundance, distribution, or ability traffic other mRNAs. Conversely, protrusions directed used mass spectrometry identify binding partners endogenous KIF1C, revealed dramatic dysregulation number identity interactors response mislocalization. These results therefore uncovered mechanistic connection between specificity protein–protein interactions. anticipate mechanism limited likely be general principle impacts functions proteins protrusion-enriched cells.

Language: Английский

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