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MicroRNA sequence codes for small extracellular vesicle release and cellular retention

Subjects

Abstract

Exosomes and other small extracellular vesicles (sEVs) provide a unique mode of cell-to-cell communication in which microRNAs (miRNAs) produced and released from one cell are taken up by cells at a distance where they can enact changes in gene expression1,2,3. However, the mechanism by which miRNAs are sorted into exosomes/sEVs or retained in cells remains largely unknown. Here we demonstrate that miRNAs possess sorting sequences that determine their secretion in sEVs (EXOmotifs) or cellular retention (CELLmotifs) and that different cell types, including white and brown adipocytes, endothelium, liver and muscle, make preferential use of specific sorting sequences, thus defining the sEV miRNA profile of that cell type. Insertion or deletion of these CELLmotifs or EXOmotifs in a miRNA increases or decreases retention in the cell of production or secretion into exosomes/sEVs. Two RNA-binding proteins, Alyref and Fus, are involved in the export of miRNAs carrying one of the strongest EXOmotifs, CGGGAG. Increased miRNA delivery mediated by EXOmotifs leads to enhanced inhibition of target genes in distant cells. Thus, this miRNA code not only provides important insights that link circulating exosomal miRNAs to tissues of origin, but also provides an approach for improved targeting in RNA-mediated therapies.

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Fig. 1: Cell-type-specific miRNAs in sEVs and cells and selectivity of sEV versus cellular distribution for certain miRNAs.
Fig. 2: Motifs over-represented in miRNAs preferentially sorted into exosomes/sEVs (EXOmotifs) or retained in cells (CELLmotifs) for each cell type.
Fig. 3: CELLmotifs and EXOmotifs regulate miRNA distribution, and Alyref and Fus participate in sorting of miRNAs containing CNGGNG-type EXOmotifs.
Fig. 4: Incorporation of EXOmotifs enhances miRNA delivery and target inhibition.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. The miRNA profiling raw dataset can be found in Supplementary Table 1 as negative ΔCt values normalized by the average Ct of each corresponding sample. Average normalized raw values for each cell type and the statistics for each miRNA in cell bodies and exosomes/sEVs are shown in Supplementary Tables 2 and 4, respectively. Significance tables for comparisons between each cell type and the other four cell types in cell bodies and sEVs can be found in Supplementary Tables 3 and 5, respectively. An sEV and cellular enrichment table for each miRNA can be found in Supplementary Table 6. The primary hepatocyte miRNA profile is shown in Supplementary Table 7. The miRNA profile from the ultracentrifugation + size-exclusion chromatography experiment is shown in Supplementary Table 8. smRNA-seq data can be found in Supplementary Table 10. Pre-miRNA sequences of the expressed miRNAs used in this study can be found in Supplementary Table 11, and their mature forms are displayed in Extended Data Figs. 79. The Ensembl and miRBase databases are publicly available at http://www.ensembl.org and http://www.mirbase.org, respectively. Experimental procedures have also been uploaded to EV-TRACK42 with reference nos EV200052 and EV210287. The mass spectrometry analysis can be found in Supplementary Table 9, and the raw data have been deposited with MassIVE ID MSV000086780 and are also available at ProteomeXchange with ID PXD023895. Additional mass spectrometry details from DIA and DDA acquisitions, such as protein identification and quantification details, are available at the repositories (including all generated Spectronaut and ProteinPilot search engine files).

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Acknowledgements

This work was supported by an NIH grant (R01DK082659 to C.R.K.). R.G.-M. was supported by a Deutsche Forschungsgemeinschaft fellowship (GA 2426/1-1, Germany). G.W. was supported by a T32 grant (DK007260). T.M.Z. was supported by a FAPESP fellowship (2017/10179-9, Brazil). We also received support from the Joslin DRC (P30DK036836). S.S., S.K.P. and B.S. were supported by Shared Instrumentation Grant S10 OD016281to the Buck Institute. S.K.P. was supported by a fellowship from the Glenn Foundation. We thank A.M. Ciulla and the BPF Genomics Core Facility at Harvard Medical School for their expertise and instrument availability that supported this work. We thank M. Ericsson from the Electron Microscopy Facility at the Harvard Medical School for assistance with electron microscopy experiments. We thank J.M. Dreyfuss and H. Pan from the Joslin Bioinformatics and Biostatistics Core for assistance with data analysis and A. Wood from the Joslin Flow Cytometry Core for assistance with experiments.

Author information

Authors and Affiliations

Authors

Contributions

R.G.-M. designed research, performed experiments, analysed the data shown in Figs. 14 and Extended Data Figs. 110 and wrote the manuscript. G.W. helped with vector generation for the miRNA overexpression shown in Fig. 3a–f and Extended Data Figs. 79. B.B.B. helped with the size-exclusion chromatography experiments shown in Extended Data Fig. 6. T.M.Z. helped with analysis of the miRNA profiling and motifs shown in Figs. 1 and 2. S.S., S.K.P. and B.S. performed the proteomic study and its analysis shown in Fig.3g–j and Extended Data Fig. 10a–c. C.R.K. designed the research, wrote the manuscript and supervised the project.

Corresponding author

Correspondence to C. Ronald Kahn.

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A patent submitted by R.G.-M. and C.R.K. (US patent no. US2021171949A1) covers the data and applications of the findings described in this manuscript.

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Extended data figures and tables

Extended Data Fig. 1 General features of sEV secretion among the five cell types studied.

a) Number of vesicles over 48 h released by each cell type normalized by the number of cells in the tissue-culture plate (n=4). *P≤0.05 (indicated cell type versus all other cell types), § P≤0.05 (indicated cell type versus 3T3-L1, C2C12 and SVEC) (Kruskal-Wallis followed by Mann-Whitney U test). b) Average vesicle size of the sEV as determined by Nanoparticle tracking analysis (NTA) for each cell type (n=4). c) Average size distribution and number of vesicles released per cell for each of the five cell types. d) Immunoblotting for the indicated sEV (ALIX, TSG101 and CD9) and cellular (GM130, CANX) markers in sEV and cell lysates from AML12 hepatocytes and BAT brown adipocytes. e) Electron micrograph showing CD63 gold immunostaining of sEV isolated from C2C12 cells. f) RNA yield obtained from sEV isolated from each cell type and normalized by the number of cells in the tissue-culture plate (n=3). *P≤0.05 (indicated cell type versus all other cell types); § P≤0.05 (indicated cell type versus 3T3-L1 and BAT) (Kruskal-Wallis followed by Mann-Whitney U test). g) Principal component analysis showing cellular miRNA profiles for each cell type. h) Heatmap showing the top 10 representative cellular miRNAs of each cell type. i) Heatmap showing the top 10 representative sEV miRNAs of each cell type. j) Comparative miRNA profile between cell-derived sEV and non-conditioned medium (NCM). Same volume of NCM as in cell-conditioned medium was processed for sEV isolation by differential ultracentrifugation. RNA was isolated and a miRNA profiling was performed for NCM. The miRNA expressions for the 13 miRNAs found sEV-enriched in all 5 cell types were compared to the NCM average Ct by ΔΔct method and represented as fold change. Each dot is the relative average value of each of the five cell types. Data are expressed as mean ± SEM.

Extended Data Fig. 2 Cell-type specific sEV enrichment of miRNAs and sEV versus cell housekeeping miRNAs. Effect of percentage of CG and Gibbs free energy (ΔG) in miRNA sorting or cellular retention.

a) Normalized gene expression of representative miRNAs showing cell-type specific sEV enrichment: miR-696 (BAT), miR-770-5p (C2C12), miR-1927 (3T3-L1), miR-1931 (AML12) and miR-718 (SVEC). Average Ct from the whole miRNA profile was used for normalization for each sample (n=3-4). *P≤0.05 (Limma t-test). b) Normalized gene expression of two representative miRNAs (miR-138-5p and miR-501-5p) showing similar sEV and cellular expression for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample (n=3-4). c) Percentage of CG content in the sequence of the miRNAs: those sorted into the sEV in all cells are shown in red (n=13); those sorted into sEV in 3 or 4 of the five cell types are shown in green (n=90); those not enriched in either sEV or cells are shown in black (n=109); those retained in 3 or 4 cell types are shown in pink (n=97); and those retained in the cell bodies of all cell types are shown in blue (n=43). d) The calculated Gibbs free energy (ΔG) for each of the miRNA in each of the five groups described in panel c. For c, d lines indicate mean value, **P≤0.01, *** P≤0.001 between the indicated group and the other four groups (ANOVA followed by Bonferroni post-hoc test in c, d). Data are expressed as mean ± SEM.

Extended Data Fig. 3 Comparison of all identified Extended and Core EXOmotifs (a) and CELLmotifs (b) among the five different cell types.

a) Fold enrichment (left half) and abundance (right half, as percentage of sEV-enriched miRNAs) containing the sEV-associated miRNA motifs. The first column indicates the cell type where these motifs were originally identified (see Main Fig. 2a) and here shown in its predominant form in the second column (Extended motifs) and fifth column (Core motifs). The fold enrichment is shown in a red (high sEV enrichment)-white (neutral)-blue (cell enrichment) color gradient for indicated cell types displayed below. The abundance is shown in a red (high)-white (low) color gradient. The rectangles highlight the enrichment and presence of the motifs in the cell types where they were originally identified. b) Fold enrichment (left half) and abundance (right half, as percentage of cell-enriched miRNAs) containing the Cell-associated miRNA motifs. As in a), the first column indicates the cell type where these motifs were originally identified (see Main Fig. 2b) and here shown in its predominant form in the second column (Extended motifs) and fifth column (Core motifs). The fold enrichment is shown in a blue (high cell enrichment)-white (neutral)-blue (red sEV enrichment) color gradient for indicated cell types displayed below. The abundance is shown in a blue (high)-white (low) color gradient. The rectangles highlight the enrichment and presence of the motifs in the cell types where they were originally identified.

Extended Data Fig. 4 Location of EXO and CELLmotifs and comparison between small miRNAseq (smRNAseq) and qPCR-based miRNA profiling.

a, b) Percentage of miRNAs showing indicated EXOmotifs (a) or CELLmotifs (b) either in the 5′ half (nucleotides 1-9, light yellow bars) or 3′ half (from nucleotide 10 to the 3′ end, orange bars) of the miRNA sequence. c) sEV and cell lysates from differentiated 3T3-L1 white adipocytes and AML12 hepatocytes were subjected to smRNAseq or qPCR-based profiling (n=4 for each cell type and compartment). Detected miRNAs by each method in each cell type correspond to the sum of black (non-selectively distributed miRNAs) and blue bars (selectively distributed miRNAs either in sEV-enriched or cell-enriched). The percentages in the blue bars refer to the ratio between the number of selectively distributed miRNA and the total number of detected miRNAs for each method and cell type. d) Venn diagrams indicating the number of miRNAs with a selective distribution in sEV or cells detected by smRNAseq (blue circles) and qPCR (green circles) in 3T3-L1 (above) or AML12 (below). The total number of miRNAs detected simultaneously by these two methods was 180. e) Table depicting the top EXOmotif found by HOMER software in sEV-enriched miRNAs from 3T3-L1 and AML12 detected by smRNAseq. Fold enrichment refers to the ratio between presence in the sEV-enriched miRNAs and presence in the rest of miRNAs (background).

Extended Data Fig. 5 Comparison of miRNA profiling of AML12 and primary hepatocytes.

Primary hepatocytes were isolated from C57Bl/6J wild-type mice (n=4) and cultured for 48 h in exosome-free medium to collect sEV and cell lysates, which were later subjected to RNA isolation and miRNA profiling for comparison to AML12 hepatocytes. a) Number of miRNAs showing selective cellular retention, non-selective distribution or selective sEV sorting in AML12 and primary hepatocytes. The selective distribution column is the sum of sEV- and cell-enriched miRNAs divided by the total number of miRNAs detected as percentage. FDR < 0.1. b) Venn-diagram showing the number of sEV- (red) and cell-enriched miRNAs (blue) in AML12 and primary hepatocytes and the overlap between them. c, d) Motifs associated to sEV (c) and cell (d) enrichment in primary hepatocytes. The table shows the significance of the enrichment (P-value), false discovery rate (FDR), the percentage of miRNAs significantly enriched in sEV (in c) or cell (in d) that contain the motif, the percentage of miRNAs not enriched in the background miRNAs containing the motif and the fold-enrichment as the ratio between the previous two columns.

Extended Data Fig. 6 Isolation of sEV and NV using an additional step of size exclusion chromatography.

a) Diagram of the isolation method used to obtain cellular, sEV-p100 pellet, sEV-SEC and NV-SEC samples using two rounds of ultracentrifugation followed by size exclusion chromatography (SEC). (n=4). b) NTA analysis for EV concentration (top graph) of the 30 fractions obtained from SEC. These fractions were pooled in pairs in some cases and concentrated using Amicon centrifugal 3 KDa filter prior assessing protein concentration (bottom graph). c) Immunoblot for classical exosomal markers CD63 and CD9 for the concentrated fractions shown in b, bottom graph. d) PCA plot for the miRNA profile from the cells, sEV-p100, sEV-SEC and NV-SEC samples. e) Heatmap of the top differentially expressed miRNAs among sEV-p100, sEV-SEC and NV-SEC. High expression is shown in red and low expression in blue. f) Pearson correlation between averaged normalized miRNA expression levels in sEV-p100 and sEV-SEC. Expression levels were first normalized to average ct of each sample. g) Motifs found overrepresented in sEV-SEC enriched-miRNAs compared to cellular-enriched miRNAs.

Extended Data Fig. 7 Additional information about mutations in miR-431-5p, miR-140-3p and miR-677-5p.

a) Table depicting the name and sequence of the CELLmotif AGAAC incorporated in miR-431-5p. Bold underlying text in the sequence indicated changed nucleotides in the guide strand of the miRNA used to introduce the CELLmotif AGAAC. Nucleotides in the passenger strand were also modified to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for the constructs shown in a. Red means high probability of pairing, while blue indicates low probability calculated by RNAfold WebServer software. Arrow indicates the location of the mutated nucleotides. c) Normalized gene expression for miR-431-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. d) Table depicting the name and sequence of the wild-type miR-140-3p and mutated version without CELLmotif AGAAC. Bold underlying text in the sequence indicated changed nucleotides in the guide strand of the miRNA used to remove the CELLmotif AGAAC. Nucleotides in the passenger strand were also modified to maintain miRNA structure. e) Predicted structure for the hairpin miRNA for the constructs shown in d. Red means high probability of pairing, while blue indicates low probability. Arrow indicates the location of the mutated nucleotides. f) Normalized gene expression for miR-140-3p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. g) Table showing the name and sequence of the wild-type miR-677-5p and the version in which CELLmotifs were mutated. Bold underlined text in the sequence indicated mutated nucleotides in the guide strand of the miRNA to remove CELLmotifs CAGU and AUU[A/G]. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. h) Predicted structure for the hairpin miRNA for the constructs shown in g. Red means high probability of pairing while blue indicates low probability. Arrows indicate the location of the mutated nucleotides. i) Normalized gene expression for miR-677-5p wild-type in cells and exosomes/sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. Data are expressed as mean ± SEM. n=3-4. *P≤0.05 (Limma t-test).

Extended Data Fig. 8 Additional information about mutations in miR-34c.

a) Table depicting the name and sequence of the different EXOmotifs introduced in miR-34c-5p. Bold underlying text in the sequence indicates changed nucleotides in the guide strand of the miRNA. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for each of the constructs shown in a. Red means high probability of pairing while blue indicates low probability calculated by RNAfold WebServer software. Arrows indicate the location of the mutated nucleotides. c) Normalized expression for miR-34c-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. *P≤0.05 (Limma t-test). d) To visualize the changes in the sEV and cell content of each miRNA construct before sEV enrichment calculation, normalized expression of miR-34c wild-type (WT) or its EXOmotif-containing versions miR-34-UGUGU, miR-34-CAUG and miR-34-CGGGAG are displayed for the cells overexpressing each of the miR-34c versions. miR-138-5p was used to normalize expression in sEV versus cells as we previously showed that the levels of this miRNA are equivalent in both compartments.*P≤0.05 (Mann-Whitney U test comparing sEV and cell expression for the same miRNA version, i.e. red versus blue bars). e) Absolute copy number of the miRNAs displayed in the x-axis overexpressed in brown adipocytes were quantified in sEV isolated by ultracentrifugation followed by size exclusion chromatography and normalized by the miRNA copy number in all producing cells for each sample. f) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from AML12 hepatocytes. g) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from SVEC endothelial cells. In both panels f, g, the dashed line separates preferential sEV enrichment (above line) versus preferential cellular enrichment (below line). Expression was normalized to the expression of miR-501-5p, which is to be equally abundant in sEV and cells for each cell type. Data are expressed as mean ± SEM. n=3, *P≤0.05, Kruskal-Wallis followed by Mann-Whitney U tests.

Extended Data Fig. 9 Additional information about mutations in miR-26a.

a) Table depicting the name and sequence of the different EXOmotifs introduced in miR-26a-5p. Bold underlying text in the sequence indicates changed nucleotide/s in the guide strand of the miRNA. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for each of the constructs shown in a. Red means high probability of pairing while blue indicates low probability calculated by RNAfold WebServer software. Arrows indicate the location of the mutated nucleotide/s. c) Normalized expression for miR-26a-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. *P≤0.05 (Limma t-test). d) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from SVEC endothelial cells. The dashed line separates preferential sEV enrichment (above line) versus preferential cellular enrichment (below line). Expression was normalized to the expression of miR-501-5p. *P≤0.05 (Kruskal-Wallis followed by Mann-Whitney U test), n=3-4.

Extended Data Fig. 10 Further information miRNA pulldown and direct transfection of AML12 cells.

a) Analyses for Molecular Function (left) and Cellular Component Gene Ontology (right) of the 67 proteins identified in the proteomic study. b) Table showing average values for relative binding enrichment of the proteins listed in the first column to the miRNA constructs shown in the top row. The columns 2-5 refer to miR-34c and its CGGGAG-containing version, while columns 6-8 refer to miR-26a and its CGGGAG-containing version. In both cases, binding to wild-type miRNAs was set as 1, and the binding of the other miRNA constructs (scramble and CGGGAG-containing version) was normalized respect to that. Only those proteins showing a log2 Fold Enrichment of CGGGAG-containing miRNA version versus wild-type miRNA >3 (>8 fold) were included. c) Brown adipocytes overexpressing wild type miR-34c (OE-miR-34c-5p-WT, grey bars) or CGGGAG-containing miR-34c (OE-miR-34c-5p-CGGGAG, red bars) were transfected with either control siRNA, Alyref siRNA or Fus siRNA (as indicated in x-axes) and analyzed by qPCR for knockdown efficiency for Alyref (left graph) or Fus (right graph), or by immunoblotting (right). *P≤0.05 (Kruskal-Wallis followed by Mann-Whitney U test), n=3. d) EXOmotif-containing miR-34c versions have the same efficiency in reducing target gene expression than wild-type miR-34c. AML12 hepatocytes were directly transfected with mimic miR-34c wild-type or its mutant versions miR-34-UGUGU, miR-34-CAUG and miR-34-CGGGAG or non-targeting miRNA (control) for 24 h and expression of predicted and experimentally-validated miR-34c target genes were analyzed. TATA-box binding protein (Tbp) was used as housekeeping gene. n=6. *P≤0.05, ***P≤0.001 (ANOVA followed by Bonferroni post-hoc test). Data are expressed as mean ± SEM.

Supplementary information

Supplementary Fig. 1

This file contains uncropped gels for the images shown in Extended Data Figs. 1, 6 and 10.

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Supplementary Information

This file contains the legends for Supplementary Tables 1–11.

Supplementary Table 1

Negative ΔCt value for each miRNA normalized by average Ct in sEVs/exosomes and cell bodies obtained from miRNA profiling.

Supplementary Table 2

Average normalized raw expression and statistics of each miRNA in cell bodies for each cell type.

Supplementary Table 3

Significance table of cell-type-specific miRNAs for each cell type.

Supplementary Table 4

Average normalized raw expression and statistics of each miRNA in exosomes/sEVs for each cell type.

Supplementary Table 5

Significance table of exosome/sEV-specific miRNAs for each cell type.

Supplementary Table 6

sEV enrichment miRNAs.

Supplementary Table 7

Primary hepatocyte miRNA profile.

Supplementary Table 8

Ultracentrifugation + SEC miRNA profile.

Supplementary Table 9

List of proteins from the miRNA pulldown experiment and their statistics.

Supplementary Table 10

Expression (as counts per million, CPM) of each detected miRNA in the smRNA-seq experiment.

Supplementary Table 11

Pre-miRNA sequences showing all mutations in both guide and passenger strands.

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Garcia-Martin, R., Wang, G., Brandão, B.B. et al. MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature 601, 446–451 (2022). https://doi.org/10.1038/s41586-021-04234-3

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