Axonal precursor mi RNA s hitchhike on endosomes and locally regulate the development of neural circuits
2020; Springer Nature; Volume: 39; Issue: 6 Linguagem: Inglês
10.15252/embj.2019102513
ISSN1460-2075
AutoresEloina Corradi, Irene Dalla Costa, Antoneta Gavoci, Archana Iyer, Michela Roccuzzo, Tegan A. Otto, Eleonora Oliani, Simone Bridi, Stephanie Strohbuecker, Gabriela Santos‐Rodriguez, Donatella Valdembri, Guido Serini, Cei Abreu‐Goodger, Marie‐Laure Baudet,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoArticle19 February 2020Open Access Source DataTransparent process Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits Eloina Corradi Eloina Corradi Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Irene Dalla Costa Irene Dalla Costa Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Antoneta Gavoci Antoneta Gavoci Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Archana Iyer Archana Iyer Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Michela Roccuzzo Michela Roccuzzo Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Tegan A Otto Tegan A Otto Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Eleonora Oliani Eleonora Oliani Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Simone Bridi Simone Bridi Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Stephanie Strohbuecker Stephanie Strohbuecker Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Gabriela Santos-Rodriguez Gabriela Santos-Rodriguez Unidad de Genómica Avanzada (Langebio), Irapuato, Mexico Search for more papers by this author Donatella Valdembri Donatella Valdembri Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Search for more papers by this author Guido Serini Guido Serini orcid.org/0000-0002-3502-8367 Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Search for more papers by this author Cei Abreu-Goodger Cei Abreu-Goodger Unidad de Genómica Avanzada (Langebio), Irapuato, Mexico Search for more papers by this author Marie-Laure Baudet Corresponding Author Marie-Laure Baudet [email protected] orcid.org/0000-0001-8957-5545 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Eloina Corradi Eloina Corradi Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Irene Dalla Costa Irene Dalla Costa Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Antoneta Gavoci Antoneta Gavoci Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Archana Iyer Archana Iyer Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Michela Roccuzzo Michela Roccuzzo Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Tegan A Otto Tegan A Otto Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Eleonora Oliani Eleonora Oliani Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Simone Bridi Simone Bridi Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Stephanie Strohbuecker Stephanie Strohbuecker Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Gabriela Santos-Rodriguez Gabriela Santos-Rodriguez Unidad de Genómica Avanzada (Langebio), Irapuato, Mexico Search for more papers by this author Donatella Valdembri Donatella Valdembri Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Search for more papers by this author Guido Serini Guido Serini orcid.org/0000-0002-3502-8367 Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Search for more papers by this author Cei Abreu-Goodger Cei Abreu-Goodger Unidad de Genómica Avanzada (Langebio), Irapuato, Mexico Search for more papers by this author Marie-Laure Baudet Corresponding Author Marie-Laure Baudet [email protected] orcid.org/0000-0001-8957-5545 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Author Information Eloina Corradi1, Irene Dalla Costa1,‡, Antoneta Gavoci1,‡, Archana Iyer1,‡, Michela Roccuzzo1,‡, Tegan A Otto1, Eleonora Oliani1, Simone Bridi1, Stephanie Strohbuecker1, Gabriela Santos-Rodriguez2, Donatella Valdembri3,4, Guido Serini3,4, Cei Abreu-Goodger2 and Marie-Laure Baudet *,1 1Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy 2Unidad de Genómica Avanzada (Langebio), Irapuato, Mexico 3Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy 4Department of Oncology, University of Torino School of Medicine, Candiolo, Italy ‡These authors contributed equally to this work *Corresponding author: Tel: +39 0462 85334; E-mail: [email protected] The EMBO Journal (2020)39:e102513https://doi.org/10.15252/embj.2019102513 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 Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments. Synopsis Mechanisms regulating subcellular localization and local function of non-coding RNA are not well understood. Here, neuronal pre-miRNAs are found to traffic along axons docked to endosomes, with axonal processing of pre-miRNAs leading to the inhibition of local protein synthesis. Precursor miRNAs hitchhike onto late endosomes/lysosomes to reach the growth cone. Sema3A, but not Slit2, axon guidance cue induces local biogenesis of specific miRNAs within axons. Newly generated miRNAs inhibit basal translation of TUBB3 but not APP upon Sema3A exposure. miRNAs regulate growth cone steering and the establishment of functional connections. Introduction Most cells are polarized, with an intracellular milieu partitioned into various organelles, cytosolic and membrane microdomains that accomplish specialized biological and regulatory functions. Neurons are highly polarized, and their axons constitute a unique cellular outpost with distinctive autonomous functions. During development, axons grow and generate a complex network of interconnected neurons. To establish these connections, the tip of the growing axon, the growth cone, is guided with exquisite precision by attractant and repellent chemotropic cues en route to its target. Axons must sometimes navigate a significant distance before reaching their final destination. In large mammals, axons can even reach targets located meters away. The distance between growth cone and cell body poses a particular challenge to developing neurons, as growth cones must be able to rapidly and accurately transduce environmental information to ensure highly precise directional steering, without the immediate intervention from the soma. To overcome this challenge, growth cones store, locally produce, and fine-tune the levels of their own proteins through local protein synthesis (LPS), from a rich repertoire of mRNAs that are selectively trafficked there (Cioni et al, 2018). Overall, mRNA localization and its corollary, local translation, are key mechanisms to create and sustain polarity by conferring functional autonomy to a variety of subcellular compartments including axons (Chin & Lécuyer, 2017). Recent evidence suggests that not only mRNAs but also various additional RNA species such as the small non-coding RNAs (ncRNAs) miRNAs (Kye et al, 2007; Lugli et al, 2008; Natera-Naranjo et al, 2010), linear long ncRNAs (Cabili et al, 2015), and circular RNAs (You et al, 2015) are localized to and enriched within subcellular outposts. The mechanisms of ncRNA transport to these compartments and the biological functions of local ncRNAs have, however, remained elusive. Gaining such fundamental insight on axons is crucial, as derailed axonal transport is a common factor in several incurable neurodegenerative disorders (Liu et al, 2012). Dicer is essential for the production of most ~22nt active, mature miRNAs from short hairpin precursor miRNAs (pre-miRNAs) (Grishok et al, 2001; Hutvagner et al, 2001). Remarkably, several reports have revealed the presence of Dicer within growth cones (Hengst, 2006; Aschrafi et al, 2008; Zhang et al, 2013; Hancock et al, 2014; Kim et al, 2015; Vargas et al, 2016; Gershoni-Emek et al, 2018). These findings raise the intriguing possibility that inactive pre-miRNAs are trafficked along axons to the growth cone and locally activated to exert their function. Here, we thus explore the transport mechanisms of pre-miRNAs within axons using a novel molecular beacon-based approach. We discover that pre-miRNAs exploit a vesicle-based transport system to reach the axon tip, where they are subsequently stored. We then investigate the local function of pre-miRNAs within growth cones. We uncover that upon repellent cue exposure, specific pre-miRNAs are locally processed into active miRNAs that rapidly inhibit the basal translation of a selected transcript. This, in turn, induces directional steering ex vivo and in vivo, and promotes the development of a fully functional neuronal circuit. Collectively, these results reveal that miRNAs hitchhike on vesicles to distal axons in an inactive form. At the growth cone, they are activated, on demand, to acutely inhibit basal translation of specific transcripts ultimately promoting growth cone steering and the highly accurate assembly of neuronal circuits. Results Molecular beacons are new, specific tools to detect endogenous pre-miRNAs in cells To investigate pre-miRNA transport mechanisms, we first established that these precursors are indeed located to axons. Analysis of our previously published dataset of small RNAs (Bellon et al, 2017) revealed the existence of sequences corresponding to the loop region of 42 pre-miRNAs in axons and growth cones of Xenopus laevis retinal ganglion cells (RGCs), our model system (Appendix Fig S1A). We validated, by RT–PCR, the presence of the abundant pre-miR-182, pre-miR-181a-1, and pre-miR-181a-2 in eyes, and pure axonal RNA that we collected by laser capture microdissection (LCM; Fig 1A–C and Appendix Fig S1A). We also examined the relative expression levels of the two miR-181a precursors by RT–qPCR, and found that a-1 was 5.9× significantly less abundant than a-2 in eyes (Appendix Fig S1B), yet 2.5× more abundant in isolated axons (Appendix Fig S1C). This indicates that pre-miR-181a-1 may be preferentially targeted to axons and growth cones. Figure 1. Molecular beacons are new, specific tools to detect endogenous pre-miRNAs in cells A. Schematic representation of the experimental protocol. LCM, laser capture microdissection. B, C. RT–PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and in axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; –, PCR no template control; NT, RT no template control. D. Schematic of MB, pre-miR-181a-1, and their hybridization complex. MB, molecular beacon. E. Schematic of thermal denaturation profile of the MB. MB, molecular beacon. F. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. MB, molecular beacon; Tm, MB melting temperature. G. Schematic representation of the experimental protocol. Concentrations used are as follows: 5 μM MB; 250 μM co-MO; 250 μM pri-miR-MO. MB, molecular beacon; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site. H. Quantification of the expression levels of pre-mir-181a-1 using the 2−ΔCt method and U6 as normalizer from small total RNA fraction (< ˜150 nt). Each data point corresponds to one independent experiment. n = 3 independent experiments. Values are mean ± SEM. ns, not significant. I. Total number of MB puncta normalized to axon length (μm). Each data point corresponds to one axon. Total number of puncta and axons analyzed is as follows: 928 puncta and 61 axons (WT); 226 puncta and 15 axons (co-MO); 208 puncta and 35 axons (MO). n = 4 independent experiments. Values are mean ± SEM. ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site. J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. MB, molecular beacon. Scale bars: 5 μm. K. Schematic representation of the experimental protocol. Concentrations used are as follows: 5 μM MB; 200–250 ng/μl cy3-pre-miR-181a-1. MB, molecular beacon. M. Schematic representation of cy5-labeled pre-miR-181a-1. N. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed is as follows: 354 puncta and 32 axons (MB); 337 puncta and 32 axons (cy5-pre-miR-181a-1). n = 5 independent experiments. Values are mean ± SEM. MB, molecular beacon. O. Representative image of RGC axons. White, red, and blue arrows indicate, respectively, colocalized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were normally distributed (Shapiro–Wilk test), one-way ANOVA followed by Tukey's multiple comparison post hoc test (H). Data were not normally distributed (Shapiro–Wilk test), and Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test (I). Download figure Download PowerPoint We next explored how pre-miRNAs reach the growth cone. We specifically examined the transport dynamics of endogenous pre-miR-181a-1, more abundant than pre-miR-181-a2 in axons (Appendix Fig S1C), by adapting molecular beacon (MB) technology. MBs are single-stranded oligonucleotide probes which fluoresce only when hybridized to their target (Santangelo et al, 2006) (Fig 1D). The MB backbone and sequence was carefully designed to maximize the probe's stability, signal-to-noise ratio, and specificity within the cell (see Methods for details). Thermal denaturation assay (Fig 1E) revealed increased fluorescence levels at low temperatures in the presence of in vitro transcribed pre-miR-181a-1 (Fig 1F) but not in the presence of (i) pre-miR-187 which is absent from RGCs (this study and Ref. (Bellon et al, 2017)), (ii) a modified pre-miR-181a-1 lacking the loop region, and (iii) short double-stranded miR-181 mimic (see Methods; Appendix Fig S1D–F). This confirms that our MB is able to specifically hybridize with and detect pre-miR-181a-1 in vitro. To test MB specificity ex vivo, we first examined change in MB signal when endogenous pre-miR-181a-1 is knocked down (Fig 1G). Blocking Drosha cleavage with morpholino (pri-miR-MO) led to a significant decrease in pre-miR-181a-1 levels by 56.3% in the eye (Fig 1H) and of MB puncta by 58.4% (Fig 1I and J). We did not observe significant differences between WT and co-MO (Fig 1H–J). Second, we examined whether cy3-labeled MB colocalized with exogenous cy5-labeled pre-miR-181a-1 (Fig 1L) or pre-miR-187 (Appendix Fig S1G) that we serially electroporated into eye primordia (Fig 1K and N, and Appendix Fig S1H–L). Within axons, 70.1–77.2 and 2.9% of MB puncta colocalized with pre-miR-181a-1 and pre-miR-187 puncta, respectively (Fig 1M and Appendix Fig S1L). This suggests that the MB can specifically recognize pre-miR-181a-1 ex vivo. Endogenous and exogenous pre-miRNAs are actively trafficked along axons We then characterized endogenous pre-miR-181a-1 trafficking dynamics in RGC axons by single axon live imaging and kymograph analysis following targeted electroporation of MBs into retinal cells (Fig 2A–C). MB-labeled pre-miR-181a-1 puncta were detected throughout the entire length of growing RGC axons and within the central domain of growth cones (Fig 2B). Equal frequencies of anterogradely and retrogradely moving puncta were detected (Fig 2D). Upon subdivision of the puncta population based on speed [stationary (< 0.2 μm/s), moving (0.2–0.5 μm/s), and fast-moving puncta (> 0.5 μm/s) (Maday et al, 2014; Leung et al, 2018)], we observed that the fast anterogradely moving puncta were significantly faster on average than their retrograde counterpart (Fig 2E). These observations likely reflect the properties of molecular motors: Anterograde kinesins are highly processive motors, while retrograde dyneins make frequent back and side steps (Maday et al, 2014). No significant differences in puncta directionality (Fig 2D) and velocities (Fig 2E) between endogenous and exogenous pre-miR-181a-1 were observed (Movies EV1 and EV2). Figure 2. Endogenous and exogenous pre-miRNAs are actively trafficked along axon microtubules Schematic representation of the experimental paradigm. Concentrations used are as follows: 5 μM MB; 200–250 ng/μl cy3-pre-miR-181a-1. MB, molecular beacon. Representative image of a single distal RGC axon from MB-electroporated retina. Growth cone wrist and central domain are indicated with white arrow and star, respectively. Dashed white line delineates the axon. MB, molecular beacon. Scale bars: 5 μm. Illustrative kymograph. Scale bars: 5 μm. Frequency distribution (in percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta along the RGC axon shaft. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed is as follows: 353 puncta and 20 axons (endo); 484 puncta and 29 axons (exo). n = 3 (endo) and n = 4 (exo) independent experiments. Values are mean ± SEM. MB, molecular beacon; ns, not significant. Average velocity of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta. Each data point corresponds to one punctum. Total number of puncta and axons analyzed is as follows: 353 puncta and 20 axons (endo); 484 puncta and 29 axons (exo). n = 3 (endo) and n = 4 (exo) independent experiments. Values are median with interquartile range. MB, molecular beacon; ns, not significant; antero, anterograde movement; retro, retrograde movement. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed is as follows: 67 particles and 20 axons (endo); 82 particles and 29 axons (exo). n = 3 (endo) and n = 4 (exo) independent experiment. Values are mean ± SEM. MB, molecular beacon. MSD alpha-coefficient distribution for each single MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particle. Each data point corresponds to one particle. Total number of particles and axons analyzed is as followed: 67 particles and 20 axons (endo); 82 particles and 29 axons (exo). n = 3 (endo) and n = 4 (exo) independent experiments. Values are median with interquartile range. MB, molecular beacon; ns, not significant. Relative frequency distribution (percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles. Each data point corresponds to one independent experiment. Total number of particles and axons analyzed is as follows: 67 particles and 20 axons (endo); 82 particles and 29 axons (exo). n = 3 (endo) and n = 4 (exo) independent experiments. Values are mean ± SEM. MB, molecular beacon; ns, not significant. Schematic representation of the experimental paradigm. Five micromolar MB was electroporated. MB, molecular beacon. Representative kymographs before nocodazole (top panel; −Noco) and 30 min after 2.4 μM Noco bath application (bottom panel; +Noco). Stationary puncta in both panels are indicated with black arrows. Scale bars: 5 μm. Frequency distribution (in percentage) of MB punctum speed. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed is as follows: 358 puncta and 25 axons (−Noco); 503 puncta and 34 axons (+Noco). n = 3 independent experiments. Values are mean ± SEM. MB, molecular beacon; ns, not significant; Noco, nocodazole. Frequency distribution of MB punctum speed. Each data point corresponds to one punctum. Total number of puncta and axons analyzed is as follows: 358 puncta and 25 axons (−Noco); 503 puncta and 34 axons (+Noco). n = 3 independent experiments. Values are median with interquartile range. MB, molecular beacon; ns, not significant; Noco, nocodazole. Data information: **P < 0.01, ****P < 0.0001. Two-way ANOVA followed by Sidak's multiple comparison post hoc test (D, K). Two-way ANOVA followed by Tukey's multiple comparison post hoc test (E, H). Data were not normally distributed (Shapiro–Wilk test). Two-tailed Mann–Whitney test (G, L). Download figure Download PowerPoint To address whether moving (≥ 0.2 μm/s) endogenous pre-miRNAs are driven by active transport or passive diffusion, we performed mean square displacement (MSD) analysis (see Methods for details). The MSD data were fitted with an anomalous diffusion model: MSD = Aτα + B (Eq1) (Otero et al, 2014). Trajectories were conservatively classified as actively driven (α > 1.5), diffusive (0.9 < α < 1.1), or confined (α < 0.5) (Otero et al, 2014). We obtained α = 1.78 (endogenous) for the combined trajectories of all particles (Fig 2F), suggesting that moving pre-miRNAs were overall actively trafficked along axons. By analyzing the α distribution of individual moving particles, we detected that the majority assumed an active motion (Fig 2G and H). As above, exogenous and endogenous pre-miRNAs appeared to behave similarly, since the computed α (Fig 2F) and α distribution (Fig 2G and H) were not significantly different. As expected, particle trajectory of each type of motion appeared to differ and most displayed a perfect or near perfect fitting reflecting a single-mode behavior (Fig EV1A–C). Collectively, these data suggest that the majority of anterogradely displaced pre-miRNAs are actively transported along the axon to reach the growth cone. Click here to expand this figure. Figure EV1. Pre-miRNAs are actively trafficked along microtubules Representative tracked particles. The dashed white lines delineate the axon. MB, molecular beacon. Scale bars: 10 μm. Representative tracked particles. The x-y trajectory for each motion type is represented within the axon. Note the difference in trajectory length according to the particle type. The dashed black lines delineate the axon. High spatial resolution of x, y displacement for each motion type and corresponding MSD plot. Most trajectories analyzed displayed a perfect or near-perfect fitting reflecting a single-mode behavior. Note the striking difference in trajectories according to particle type. Only a small portion of the actively moving particle trajectory is shown in the yellow inset. Scale bars: 1 μmx1μm. Mean cy3-pre-miR-181a-1 puncta speed before and 30 min after vincristine bath application. Concentration used is as follows: 0.1 μM vincristine. Each data point corresponds to one punctum. Total number of puncta and axons analyzed is as follows: 397 puncta and 15 axons (−vincristine); 257 puncta and 15 axons (+vincristine). n = 3 independent experiments. Values are median with interquartile range. Frequency distribution (in percentage) of cy3-pre-miR-181a-1 puncta per speed categories before and 30 min after vincristine bath application. Concentration used is as follows: 0.1 μM vincristine. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed is as follows: 397 puncta and 15 axons (−vincristine); 257 puncta and 15 axons (+vincristine). n = 3 independent experiments. Values are mean ± SEM. ns, not significant. Data information: ****P < 0.0001. Data were not normally distributed (Shapiro–Wilk test) (D, E). Two-tailed Mann–Whitney test (D). Two-way ANOVA followed by Sidak's multiple comparison test (E). Download figure Download PowerPoint Pre-miRNAs are trafficked along microtubules, associated primarily with late endosomes/lysosomes Next, we dissected the mechanisms mediating pre-miRNA axonal transport. As axonal-directed trafficking relies on microtubules (MTs) (Maday et al, 2014), we explored whether pre-miR-181a-1 is also transported via this cytoskeletal component using the MT-destabilizing drug nocodazole (Noco; Fig 2I). Kymograph analysis of time-lapse movies before (−) or after (+) a 30-min Noco incubation revealed a strong and significant increase in the proportion of stationary (< 0.2 μm/s) puncta (Fig 2J and K) and an overall 91.5% decrease in puncta's average velocities (from 0.2357 to 0.0199 μm/s; Fig 2L) after drug application. Bath application of vincristine, another MT-destabilizing agent (Jordan et al, 1985), led to similar results (Fig EV1D and E). This indicates that pre-miRNA transport along the axon shaft is mediated via microtubules. In neurons, mRNAs are trafficked along axons packaged within ribonucleoparticles (RNPs) (Bauer et al, 2017). Pre-miRNAs may also be dynamically trafficked within RNPs, as recent data on dendrites suggest (Bicker et al, 2013). However, mature miRNAs, miRNA-repressible mRNAs, and components of the miRNA processing machinery associate with late endosomes/multivesicular bodies and lysosomes (LE/Ly) in non-neuronal cells (Gibbings et al, 2009; Lee et al, 2009) and LE/Ly are detected in axons and growth cones (Falk et al, 2014; Konopacki et al, 2016). It is thus possible that pre-miRNAs adopt a non-canonical transport mode associated with LE/Ly within the axon shaft. Using CD63, a transmembrane protein enriched in a LE/Ly (Pols & Klumperman, 2009), as a marker, we examined whether CD63-eGFP-labeled vesicles and MB-labeled pre-miR-181a-1 puncta were co-trafficked within axons (Fig 3A). 71.4% of MB-positive moving puncta were co-transported with CD63-eGFP-labeled vesicle-like focal puncta, and an equal percentage of these moved anterogradely and retrogradely (Fig 3B–D, F and G, and Movies EV3 and EV4). This bidirectional transport is consistent with previously published studies on LE/Ly trafficking in embryonic neurons (Farías et al, 2017). Similar results were obtained when growth cones were cultured from older embryos (Appendix Fig S2A–G and Movie EV5). These percentages are likely an underestimation, as endogenous unlabeled vesicle present in these axons may mask the extent of this co-traffic. These results indicate that CD63-positive vesicles contribute, to a large extent, to pre-miRNA axonal transport. Furthermore, numerous co-trafficked puncta reached the growth cones and appeared to stall and accumulate within the organelle-rich central domain (Dent & Gertler, 2003) (star, Figs 2B and 3E and H–L, and Appendix Fig S2E and H–I), and in the perinuclear region (Fig 3H–L). Taken together, these results suggest that pre-miRNAs hitchhike on vesicles to reach the growth cone central domain where they are stored. Figure 3. Pre-miRNAs are trafficked along microtubules associated with vesicles A. Schematic of the expe
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