Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

Abstract

Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.

Figure 1. The sequence of miRNAs controls their localization into exosomes.

(a) Microarray analysis of exosomal and cellular miRNAs from human primary T lymphocytes in resting and activated (PMA and ionomycin) conditions. The panel shows the heatmap of the per-row normalized expression levels of selected miRNAs differentially expressed in cells (CL) versus exosomes (EXOs) in resting (REST) or activated conditions (ACT). (b) Upper panel: Venn diagrams based on miRNA microarray results showing that activation-induced changes in miRNA profile are not the same in T cells and their derived exosomes. Lower panel: Venn diagrams based on miRNA microarray results showing that some miRNAs are always specifically sorted into exosomes (EXOmiRNAs, left panel) or specifically retained in cells (CLmiRNAs, right panel), independently of the activation state of the T cell. (c) Cladogram showing the multiple alignment of mature sequences of 30 EXOmiRNAs (red) and 42 CLmiRNAs (blue), revealing that miRNA clustering coincides with preferential localization in exosomes or cells. (d) Over-represented motifs in EXOmiRNAs (EXOmotifs, left panels) and CLmiRNAs (CLmotifs, right panels); ZOOPS model was used. For each data set, the remaining miRNAs annotated in Ensembl was used as background. A Markov model of order 0 was assumed for the background sequences. E-value <10⁻⁴.

Figure 2. Loading of miRNAs into exosomes is controlled by short motifs.

(a) Sequences of wild-type miRNAs and their mutated versions used in this study. Mutated miR-17 has an EXOmotif instead of its characteristic CLmotif, whereas mutated miR-601 has a CLmotif instead of its EXOmotif. (b) EXO/CL ratio of each miRNA, obtained by dividing the number of copies in exosomes by the number of copies in cells. Copy number was determined using absolute quantitative PCR. Error bars represent s.d. (n=4). One-way ANOVA; P-value <0.01. (c) Left panel: EXO/CL ratio of mutated miR-17 and endogenous (wild-type) miR-17 and miR-18. Right panel: EXO/CL ratio of mutated miR-601 and endogenous (wild-type) miR-601 and miR-18a. Error bars represent s.d. (n=3). Student’s t-test; *P-value <0.05.

Figure 3. HnRNPA2B1 specifically binds EXOmiRNAs.

Exosome extracts were incubated with streptavidin beads coated with either a biotinylated EXOmiRNA (miR-198), a biotinylated CLmiRNA (miR-17) or a negative control (poly-A), and with non-coated beads (beads). The graphs represent the extracted ion chromatogram traces of the monoisotopic peaks corresponding to the indicated peptides, identified from hnRNPA2B1or from a non-specifically binding protein (nucleolin).

Figure 4. HnRNPA2B1 binds EXOmiRNAs through EXOmotifs and is involved in their loading into exosomes.

(a) FACS analysis of hnRNPA2B1 and CD81 in exosome-coupled beads. Exosomes were coupled to aldehyde-sulfate beads, permeabilized or left intact and incubated with antibodies to hnRNPA2B1 (middle panels) or CD81 (right panels) and secondary antibody. Exosome-coupled beads incubated with secondary antibody alone were used as negative controls (left panels). (b) qPCR analysis of miRNAs contained in hnRNPA2B1 immunoprecipitates from exosome lysates, showing the specific binding of miR-198 to hnRNPA2B1 in exosomes. Immunoprecipitation was performed with magnetic beads coated with anti-hnRNPA2B1 or anti-IgG1 control antibody. Data are presented relative to miR-17 content in control immunoprecipitates. Error bars represent s.d. (n=2). (c) Electrophoresis mobility shift assay showing the specific binding of miR-198 to hnRNPA2B1. Biotinylated miR-17, miR-198 or poly-A were incubated with or without purified human hnRNPA2B1 as indicated. (d) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to wild-type and mutated miR-198. Biotinylated wild-type and mutated miR-198 were incubated with or without purified human hnRNPA2B1 as indicated. Numbers represent protein concentration (ng μl⁻¹). (e) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to miR-601. (f) qPCR analysis of miR-18a and miR-198 in exosomes from control cells or cells silenced with siRNAs against hnRNPA2B1. Bars represent miRNA levels in exosomes (arbitrary units). Error bars represent s.d. (n=3). Student’s t-test; *P-value <0.001. (g) qPCR analysis of miR-18a and miR-198 levels in exosomes from control cells or cells overexpressing hnRNPA2B1-GFP. Bars represent miRNA levels in exosomes (arbitrary units). Error bars represent s.d. (n=3). Student’s t-test; *P-value<0.01.

Figure 5. hnRNPA2B1 is sumoylated in exosomes and this modification controls its binding to miRNAs.

(a) Representative western blot analysis showing hnRNPA2B1 in T cells and their exosomes. (b) Western blot analysis of hnRNPA2B1 sumoylation. HEK293T cells were co-transfected with SUMO-1 and hnRNPA2B1-GFP or GFP plasmids. GFP immunoprecipitates and total lysates were immunoblotted for SUMO-1 and/or GFP. Ab: antibody-conjugated Dynabeads without cell lysates. GFP: lysates from cells transfected with GFP and SUMO-1. hnRNPA2B1-GFP: lysates from cells transfected with hnRNPA2B1-GFP and SUMO-1. (c) Western blot analysis of hnRNPA2B1 sumoylation in T cells. hnRNPA2B1 was immunoprecipitated from Jurkat T cells and immunoblotted for SUMO-1 and hnRNPA2B1. A2B1: hnRNPA2B1; s-A2B1: sumoylated hnRNPA2B1; ns: nonspecific band. IP CONT: immunoprecipitation with control antibody. IP A2B1: immunoprecipitation with hnRNPA2B1 antibody. (d) Western blot analysis of hnRNPA2B1 molecular weight in exosomes and cells in the presence of the sumoylation inhibitor AA or vehicle (DMSO). Numbers below the lanes are the densitometry ratios of total hnRNPA2B1 to sumoylated hnRNPA2B1. (e) qPCR analysis of miRNA levels in exosomes from control or AA-treated cells. Bars represent miR-17, miR-18 and miR-198 levels (arbitrary units). Error bars represent s.d. (n=3). Students’s t test; *P-value <0.05. (f) qPCR analysis of miR-198 in hnRNPA2B1 immunoprecipitates from exosome lysates derived from control or AA-treated cells, showing decreased binding of miR-198 to hnRNPA2B1 in the presence of AA. Bars represent miR-198 levels (arbitrary units). Data are presented relative to the control condition. Error bars represent s.d. (n=3). Student’s t-test; *P-value<0.05.

Figure 6. Diagram representing the proposed mechanism of the sorting of miRNA into exosomes through binding to hnRNPA2B1.

PM: plasma membrane, MVB: multivesicular bodies, S: SUMO, A2B1: hnRNPA2B1.