Decoding leader cells in collective cancer invasion

Nature reviews. Cancer, Год журнала: 2021, Номер 21(9), С. 592 - 604

Опубликована: Июль 8, 2021

Язык: Английский

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RNA localization mechanisms transcend cell morphology

eLife, Год журнала: 2023, Номер 12

Опубликована: Март 3, 2023

RNA molecules are localized to specific subcellular regions through interactions between regulatory elements and binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind localization a given is restricted particular cell type. Here, we show that RNA/RBP regulate in one type predictably other types with vastly different morphologies. To determine transcriptome-wide spatial distributions across apicobasal axis human intestinal epithelial cells, used recently developed proximity labeling technique, Halo-seq. We found mRNAs encoding ribosomal (RP mRNAs) were strongly basal pole these cells. Using reporter transcripts single-molecule FISH, pyrimidine-rich motifs 5′ UTRs RP sufficient drive localization. Interestingly, same also neurites mouse neuronal In both types, activity this motif was dependent on it being UTR transcript, abolished upon perturbation RNA-binding protein LARP1, reduced inhibition kinesin-1. extend findings, compared RNAseq data from compartment cells projections enriched for highly similar sets RNAs, indicating broadly mechanisms may be transporting RNAs morphologically distinct locations. These findings identify first element known establish LARP1 as an regulator, demonstrate cut

Язык: Английский

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Mechanistic insights into the basis of widespread RNA localization

Nature Cell Biology, Год журнала: 2024, Номер 26(7), С. 1037 - 1046

Опубликована: Июль 1, 2024

Язык: Английский

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mRNA Targeting, Transport and Local Translation in Eukaryotic Cells: From the Classical View to a Diversity of New Concepts

Molecular Biology, Год журнала: 2021, Номер 55(4), С. 507 - 537

Опубликована: Май 30, 2021

Язык: Английский

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High-throughput identification of RNA localization elements in neuronal cells

Nucleic Acids Research, Год журнала: 2022, Номер 50(18), С. 10626 - 10642

Опубликована: Сен. 15, 2022

Abstract Hundreds of RNAs are enriched in the projections neuronal cells. For vast majority them, though, sequence elements that regulate their localization unknown. To identify RNA capable directing transcripts to neurites, we deployed a massively parallel reporter assay tested regulatory ability thousands fragments drawn from endogenous mouse 3′ UTRs. We identified peaks activity within several UTRs and found sequences derived these were both necessary sufficient for neurites human The adenosine guanosine residues. They at least tens hundreds nucleotides long as shortening two led significantly reduced activity. Using affinity purification mass spectrometry, RNA-binding protein Unk was associated with elements. Depletion cells drive indicating functional requirement trafficking. These results provide framework unbiased, high-throughput identification mechanisms govern transcript neurons.

Язык: Английский

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Regulation and outcomes of localized RNA translation

Wiley Interdisciplinary Reviews - RNA, Год журнала: 2022, Номер 13(6)

Опубликована: Фев. 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.

Язык: Английский

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Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation

Molecular Cell, Год журнала: 2023, Номер 83(13), С. 2290 - 2302.e13

Опубликована: Июнь 8, 2023

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics microtubule function. When cells sense excess tubulin, they trigger degradation the encoding mRNAs, which requires recognition nascent polypeptide by tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates decay mRNAs is unknown. Here, our biochemical structural analysis reveals that recruits poorly studied protein SCAPER to ribosome. SCAPER, turn, engages CCR4-NOT deadenylase complex through its CNOT11 subunit mRNA decay. mutants cause intellectual disability retinitis pigmentosa humans are impaired recruitment, degradation, microtubule-dependent chromosome segregation. Our findings demonstrate how a on ribosome physically linked factors via relay protein-protein interactions, providing paradigm for specificity cytoplasmic gene regulation.

Язык: 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, Год журнала: 2024, Номер 43(15), С. 3192 - 3213

Опубликована: Июнь 19, 2024

Язык: Английский

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G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

Cell Reports, Год журнала: 2025, Номер 44(2), С. 115237 - 115237

Опубликована: Фев. 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.

Язык: Английский

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

The EMBO Journal, Год журнала: 2020, Номер 39(21)

Опубликована: Сен. 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

Язык: Английский

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