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. 2017 Mar 30:7:45661.
doi: 10.1038/srep45661.

MicroRNA exocytosis by large dense-core vesicle fusion

Alican Gümürdü  1   2 , Ramazan Yildiz  1   2 , Erden Eren  1   2 , Gökhan Karakülah  1 , Turgay Ünver  1 , Şermin Genç  1   2 , Yongsoo Park  1
Affiliations

MicroRNA exocytosis by large dense-core vesicle fusion

Alican Gümürdü et al. Sci Rep. .

Abstract

Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. High purity of LDCVs.
(a) Schematic of the overlay assay for the LDCV purity. (b,c) LDCVs were incubated simultaneously with VAMP-2 antibody (green, left) and a membrane dye, DiI (red, middle). Overlay (yellow, right) indicates LDCVs (arrows). Scale bar, 2 μm. (c) High-magnification images of LDCVs stained with VAMP-2 antibody and DiI. (d) LDCV purity was presented as a percentage of LDCVs (yellow from panel b,c, n = 119) among total vesicles (red in overlay from panel b,c, n = 125). Total vesicles were collected from three independent purifications. The purity of LDCVs is 95%. (e) Size distribution of LDCVs analyzed using dynamic light scattering. Number distribution is presented as a percentage.
Figure 2
Figure 2. Next generation sequencing for miRNAs.
(a) Total RNA extracted from LDCVs was analyzed by electrophoresis on a 2% agarose gel. (b) Size distribution of the small RNAs. Values were calculated as a percentage based on results of electropherogram trace of total RNA libraries in the Supplementary Fig. 2. (c) Known miRNA and novel miRNA based on the number of miRNAs from RNA-seq. (d) Distribution of the number of miRNAs based on read counts; count per million (CPM) on a log2 scale. Number of known miRNAs in each CPM (log2) range is presented. (e,f) Classification of the number of miRNAs as high level (69 miRNAs) and low level (356 miRNAs) based on read counts; 1,000 CPM was applied for this classification after cut-off of <1log2 CPM (e). (f) High-level and low level-miRNAs were presented as a percentage of read counts, CPM. (g) Distribution of the most abundant miRNAs based on CPM.
Figure 3
Figure 3. miRNA exocytosis by vesicle fusion.
(a) Schematic for fusion assay. Fusion was monitored using a content-mixing assay in which GelGreen, a water-soluble and membrane-impermeable nucleic acid dye, was incorporated in liposomes. Interaction of miRNA stored in LDCVs with GelGreen increases its fluorescence. Plasma membrane-mimicking liposomes incorporated the stabilized Q-SNARE complex. VAMP-2 fragment is omitted for clarity. (b) Preincubation of liposomes with a soluble fragment of VAMP-2 (VAMP-21-96), inhibits SNARE-mediated fusion due to competitive inhibition. Control, basal fusion of LDCVs with liposomes that contain Q-SNARE. No Q-SNARE, no SNAREs incorporated in liposomes. Fluorescence intensity is normalized to the initial value (F0). (c) miR-375 exocytosis from PC12 cells. PC12 cells were stimulated with 50 mM KCl for 5 min. miR-375 released by PC12 cells in the absence or presence of KCl was analyzed using qRT-PCR. Quantitative analysis was presented as fold changes in expression relative to a control (no KCl). Data are mean ± SD (n = 4 biological and technical replicates). **p = 0.0026 (two-tailed unpaired Student’s t test).
Figure 4
Figure 4. miR-375 stored in LDCVs.
(a,b) Quantification of miRNA copy number per LDCV. (a) Workflow to determine miRNA copy number. Aliquots of LDCVs were counted using Nanoparticle Tracking Analysis (NTA). In parallel, the concentration of miRNA extracted from LDCVs was determined by qRT-PCR. A synthesized Caenorhabditis elegans microRNA (cel-miR-39) as the spike-in control was added to normalize the RNA extraction efficiency. (b) Copy number of miR-375 per LDCV. Line represents mean (n = 4 biological and technical replicates). (c) Schematic for confirming vesicle-incorporated miR-375. cel-miR-39 was mixed with LDCVs prior to RNase A treatment. (d) Relative levels of cel-miR-39 and miR-375 with or without RNase A were determined by qRT-PCR. Values represent fold changes in expression relative to a control (no RNase A). *p = 0.014 (two-tailed unpaired Student’s t test). (e) miR-375 from purified synaptic vesicles from mice brains was analyzed by qRT-PCR. Data in d and e are mean ± SD (n = 3 technical replicates).
Figure 5
Figure 5. A schematic summarizing miRNA exocytosis by vesicle fusion.
LDCVs contain catecholamines that include dopamine, adrenaline, and noradrenaline, but they also contain a variety of miRNAs including miR-375. miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+ stimulus. Released extracellular miRNAs regulate cell-to-cell communication by controlling gene silencing in target cells after endocytosis and by stimulating receptors or ion channels, thereby leading to cellular signaling. Our data suggest the new concept that miRNAs are stored in vesicles together with classical neurotransmitters and are released by vesicle fusion, thus contributing to cell-to-cell communication as a novel neuromodulator.
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