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Cited by 148

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Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides

Geekiyanage, Hirosha, Rayatpisheh, Shima, Wohlschlegel, James A., Brown, Robert, Ambros, Victor·2020·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2008323117PMC PMC7533700
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Abstract

Significance: MicroRNAs (miRNAs) are abundant inside cells, where they regulate gene expression posttranscriptionally, but are also found in circulation as remarkably stable extracellular constituents of body fluids such as human blood plasma and cerebrospinal fluid. We observed that these circulating miRNAs are predominantly associated with free Argonaut 2 (AGO2) complexes that are accessible to immunoprecipitation by AGO2 antibodies and are able to base-pair with complementary target RNAs, suggesting that, under normal physiological conditions, circulating miRNAs are largely contained in functional miRNA-mediated silencing complexes (miRISCs). However, we observe that, under certain pathological conditions, more miRNAs become associated with complexes other than free AGO2/miRISC, suggesting that the assortment of circulating miRNAs into distinct complexes offers a potential new dimension to miRNA-based diagnostics.

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Figures

Fig. 1. Model of circulating miRNA association with carrier complexes. A portion of circulating miRNAs are proposed to be associated with free miRISC/AGO2 complexes that are accessible for base pairing to target mimic oligonucleotides (blue enclosure), a subset of which are accessible for binding to anti-AGO2 antibodies (pink enclosure).
Fig. 1. Model of circulating miRNA association with carrier complexes. A portion of circulating miRNAs are proposed to be associated with free miRISC/AGO2 complexes that are accessible for base pairing to target mimic oligonucleotides (blue enclosure), a subset of which are accessible for binding to anti-AGO2 antibodies (pink enclosure).CC BY-NC-ND · PubMed Central
Fig. 2. Immunoprecipitation of circulating miRNA from human blood plasma. (A) Schematic showing the immunoprecipitation (IP) protocol where bead-conjugated mouse AGO2 antibodies are incubated overnight at 4 °C in blood plasma under native conditions (absence of detergent or any other additive). The beads are removed by magnetizing, and RNA extracted from the supernatant is subjected to qRT-PCR and/or small-RNA sequencing to detect the miRNAs depleted by AGO2 immunoprecipitation. The magnetized beads are processed either for RNA extraction followed by qRT-PCR and/or small-RNA sequencing to assess miRNA recovery, or for protein extraction and subsequent Western blotting using a rabbit anti-AGO2 antibody (orthogonal to the immunoprecipitation antibody). (B) Western blot probed with anti-AGO2 antibody to assess AGO2 associated with the captured beads following AGO2 immunoprecipitation. The red arrow shows that an AGO2 band at ∼97 kDa is detected in the anti-AGO2 immunoprecipitate but absent from the control IgG immunoprecipitate. (C) Coomassie staining following SDS/PAGE shows the sections that were sliced according to ranges of molecular weight and subjected to mass spectrometry (Dataset S1). (D) Summary of AGO2 peptide spectral counts as determined by tandem mass spectrometry. AGO2 was only detected in slice C (75 to 100 kDa) in immunoprecipitation using anti-AGO2. (E) qRT-PCR analysis of miR-16 levels in the plasma supernatant and beads to display depletion and recovery following immunoprecipitation of control IgG and AGO2. Relative miRNA levels are normalized to the mean miRNA levels of control IgG supernatant samples following normalization of all samples to the cel-miR-39 spike-in. Error bars represent SE of mean (SEM) derived from three or more experiments. Statistical significances were determined by Student t tests (P ≤ 0.05).
Fig. 2. Immunoprecipitation of circulating miRNA from human blood plasma. (A) Schematic showing the immunoprecipitation (IP) protocol where bead-conjugated mouse AGO2 antibodies are incubated overnight at 4 °C in blood plasma under native conditions (absence of detergent or any other additive). The beads are removed by magnetizing, and RNA extracted from the supernatant is subjected to qRT-PCR and/or small-RNA sequencing to detect the miRNAs depleted by AGO2 immunoprecipitation. The magnetized beads are processed either for RNA extraction followed by qRT-PCR and/or small-RNA sequencing to assess miRNA recovery, or for protein extraction and subsequent Western blotting using a rabbit anti-AGO2 antibody (orthogonal to the immunoprecipitation antibody). (B) Western blot probed with anti-AGO2 antibody to assess AGO2 associated with the captured beads following AGO2 immunoprecipitation. The red arrow shows that an AGO2 band at ∼97 kDa is detected in the anti-AGO2 immunoprecipitate but absent from the control IgG immunoprecipitate. (C) Coomassie staining following SDS/PAGE shows the sections that were sliced according to ranges of molecular weight and subjected to mass spectrometry (Dataset S1). (D) Summary of AGO2 peptide spectral counts as determined by tandem mass spectrometry. AGO2 was only detected in slice C (75 to 100 kDa) in immunoprecipitation using anti-AGO2. (E) qRT-PCR analysis of miR-16 levels in the plasma supernatant and beads to display depletion and recovery following immunoprecipitation of control IgG and AGO2. Relative miRNA levels are normalized to the mean miRNA levels of control IgG supernatant samples following normalization of all samples to the cel-miR-39 spike-in. Error bars represent SE of mean (SEM) derived from three or more experiments. Statistical significances were determined by Student t tests (P ≤ 0.05).CC BY-NC-ND · PubMed Central
Fig. 3. Plasma miRNAs can be depleted by AGO2 immunoprecipitation. (A) Small-RNA sequencing of RNAs extracted from plasma supernatant following control IgG (x-axis; n = 2) and AGO2 (y-axis; n = 2) immunoprecipitation. A cel-miR-39 spike-in was included to normalize the data to the original sample volume, allowing a direct comparison of the yields of miRNAs from control and experimental samples. Essentially all of the miRNAs detected in the sequencing dataset are below the diagonal line, indicating a general depletion of miRNAs from plasma by AGO2 immunoprecipitation. (B) Small RNA sequencing analysis of depletion vs. recovery in plasma. Depletion was calculated by subtracting AGO2 immunoprecipitated supernatant miRNA counts from control IgG immunoprecipitated supernatant miRNA counts, and recovery was calculated by subtracting control IgG-immunoprecipitated miRNA counts from the AGO2-immunoprecipitated miRNA counts. (C) Distribution of the percent depletions by anti-AGO2 immunoprecipitation for 323 miRNAs (n = 2 samples). Average depletion was ∼78% (gray dashed line); 19 of the 323 miRNAs were depleted less than 50% (blue dashed line). The positions of miR-16 and let-7a are labeled.
Fig. 3. Plasma miRNAs can be depleted by AGO2 immunoprecipitation. (A) Small-RNA sequencing of RNAs extracted from plasma supernatant following control IgG (x-axis; n = 2) and AGO2 (y-axis; n = 2) immunoprecipitation. A cel-miR-39 spike-in was included to normalize the data to the original sample volume, allowing a direct comparison of the yields of miRNAs from control and experimental samples. Essentially all of the miRNAs detected in the sequencing dataset are below the diagonal line, indicating a general depletion of miRNAs from plasma by AGO2 immunoprecipitation. (B) Small RNA sequencing analysis of depletion vs. recovery in plasma. Depletion was calculated by subtracting AGO2 immunoprecipitated supernatant miRNA counts from control IgG immunoprecipitated supernatant miRNA counts, and recovery was calculated by subtracting control IgG-immunoprecipitated miRNA counts from the AGO2-immunoprecipitated miRNA counts. (C) Distribution of the percent depletions by anti-AGO2 immunoprecipitation for 323 miRNAs (n = 2 samples). Average depletion was ∼78% (gray dashed line); 19 of the 323 miRNAs were depleted less than 50% (blue dashed line). The positions of miR-16 and let-7a are labeled.CC BY-NC-ND · PubMed Central
Fig. 4. The profiles of miRNAs depleted by AGO2 immunoprecipitation from healthy CSF samples and CSF from patients with ALS or ICP. (A) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 3) and AGO2 (y-axis; n = 3) immunoprecipitation in CSF from healthy individuals. Almost all miRNAs are below the diagonal line, indicating a general depletion of almost all miRNAs in CSF from healthy individuals. (B) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 3) and AGO2 (y-axis; n = 3) immunoprecipitation in CSF from ALS patients. Fewer miRNAs (than in A) are below the diagonal line for the ALS samples, indicating an increase in miRNAs refractory to immunoprecipitation in the CSF from ALS patients compared with healthy individuals. (C) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 2) and AGO2 (y-axis; n = 2) immunoprecipitation in CSF from patients with ICP. Again, fewer miRNAs (than in A) are below the diagonal line for the ICP samples, indicating an increase in miRNAs refractory to immunoprecipitation in the CSF from ICP patients compared with healthy individuals. For A–C, the red dots represent the miRNAs that are refractory to depletion (P ≤ 0.05) in ALS CSF (B) but are efficiently depleted from control CSF (P ≤ 0.05 or P ≤ 0.1; A); the turquoise dots represent the miRNAs that are efficiently depleted (P ≤ 0.05) in healthy control CSF (A) but refractory to depletion (P ≤ 0.05) by AGO2 immunoprecipitation in CSF from ICP patients (C). miR-203b-5p is refractory to depletion in both ALS and ICP CSF samples compared with CSF samples from healthy individuals, and thus represented in an underlined (turquoise) red dot in A. (D–F) Distribution of miRNA depletion by AGO2 immunoprecipitation in healthy control CSF (D), ALS CSF (E), and ICP CSF (F). The average depletions (dashed gray lines) are ∼62%, ∼45%, and 48%, respectively. The UMI count for each sample is normalized to the spike-in cel-miR-39.
Fig. 4. The profiles of miRNAs depleted by AGO2 immunoprecipitation from healthy CSF samples and CSF from patients with ALS or ICP. (A) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 3) and AGO2 (y-axis; n = 3) immunoprecipitation in CSF from healthy individuals. Almost all miRNAs are below the diagonal line, indicating a general depletion of almost all miRNAs in CSF from healthy individuals. (B) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 3) and AGO2 (y-axis; n = 3) immunoprecipitation in CSF from ALS patients. Fewer miRNAs (than in A) are below the diagonal line for the ALS samples, indicating an increase in miRNAs refractory to immunoprecipitation in the CSF from ALS patients compared with healthy individuals. (C) Small-RNA sequencing of supernatant following control IgG (x-axis; n = 2) and AGO2 (y-axis; n = 2) immunoprecipitation in CSF from patients with ICP. Again, fewer miRNAs (than in A) are below the diagonal line for the ICP samples, indicating an increase in miRNAs refractory to immunoprecipitation in the CSF from ICP patients compared with healthy individuals. For A–C, the red dots represent the miRNAs that are refractory to depletion (P ≤ 0.05) in ALS CSF (B) but are efficiently depleted from control CSF (P ≤ 0.05 or P ≤ 0.1; A); the turquoise dots represent the miRNAs that are efficiently depleted (P ≤ 0.05) in healthy control CSF (A) but refractory to depletion (P ≤ 0.05) by AGO2 immunoprecipitation in CSF from ICP patients (C). miR-203b-5p is refractory to depletion in both ALS and ICP CSF samples compared with CSF samples from healthy individuals, and thus represented in an underlined (turquoise) red dot in A. (D–F) Distribution of miRNA depletion by AGO2 immunoprecipitation in healthy control CSF (D), ALS CSF (E), and ICP CSF (F). The average depletions (dashed gray lines) are ∼62%, ∼45%, and 48%, respectively. The UMI count for each sample is normalized to the spike-in cel-miR-39.CC BY-NC-ND · PubMed Central
Fig. 5. Target mimic oligonucleotides can base-pair with plasma miRNAs, capturing the cognate miRNA, along with AGO2. (A) Sequence complementarity of miR-16 and let-7a target mimic oligonucleotides to their cognate miRNAs. There are five extra nucleotide overhangs on the 5′ and 3′ ends of each of the target mimic oligonucleotides. (B) Schematic showing the affinity matrix-linked target mimic assay where the biotinylated target mimic is conjugated to streptavidin beads and incubated with native plasma, without detergent or additives, for 15 min at room temperature. The beads were removed by magnetizing, and the supernatant and the magnetized beads were processed for RNA extraction or protein gel electrophoresis and Western blotting using anti-AGO2 antibody. (C) qRT-PCR analysis of the plasma supernatant following the incubation with a target mimic oligo against miR-16 or let-7a. miR-16 was depleted from plasma with a target oligo against miR-16, whereas let-7a was not depleted.. Let-7a was depleted from plasma with a target oligo against let-7a, while miR-16 was not depleted. (D) qRT-PCR analysis of the recovery of plasma miR-16 following incubation with a target mimic fully complementary to miR-16 or a scrambled target mimic. Materials and Methods and SI Appendix, Fig. S4, describe the protocol for recovering miRNAs from the target mimic capture beads. “No plasma control” represents incubation of bead-conjugated target mimics in PBS. Relative expression levels shown are normalized to the spike-in cel-miR-39 and to the scrambled target mimic-incubated plasma supernatant. Error bars represent SE of mean (SEM) derived from three or more experiments. Statistical significances were determined by Student t tests. (E) Western blot probed with anti-AGO2 antibody to assess the AGO recovered from the miR-16 and scrambled target mimic-conjugated beads following incubation in plasma. The red arrow shows the AGO2-specific band at ∼97 kDa present in the sample from the miR-16 target oligo beads and absent from the scrambled oligo beads.
Fig. 5. Target mimic oligonucleotides can base-pair with plasma miRNAs, capturing the cognate miRNA, along with AGO2. (A) Sequence complementarity of miR-16 and let-7a target mimic oligonucleotides to their cognate miRNAs. There are five extra nucleotide overhangs on the 5′ and 3′ ends of each of the target mimic oligonucleotides. (B) Schematic showing the affinity matrix-linked target mimic assay where the biotinylated target mimic is conjugated to streptavidin beads and incubated with native plasma, without detergent or additives, for 15 min at room temperature. The beads were removed by magnetizing, and the supernatant and the magnetized beads were processed for RNA extraction or protein gel electrophoresis and Western blotting using anti-AGO2 antibody. (C) qRT-PCR analysis of the plasma supernatant following the incubation with a target mimic oligo against miR-16 or let-7a. miR-16 was depleted from plasma with a target oligo against miR-16, whereas let-7a was not depleted.. Let-7a was depleted from plasma with a target oligo against let-7a, while miR-16 was not depleted. (D) qRT-PCR analysis of the recovery of plasma miR-16 following incubation with a target mimic fully complementary to miR-16 or a scrambled target mimic. Materials and Methods and SI Appendix, Fig. S4, describe the protocol for recovering miRNAs from the target mimic capture beads. “No plasma control” represents incubation of bead-conjugated target mimics in PBS. Relative expression levels shown are normalized to the spike-in cel-miR-39 and to the scrambled target mimic-incubated plasma supernatant. Error bars represent SE of mean (SEM) derived from three or more experiments. Statistical significances were determined by Student t tests. (E) Western blot probed with anti-AGO2 antibody to assess the AGO recovered from the miR-16 and scrambled target mimic-conjugated beads following incubation in plasma. The red arrow shows the AGO2-specific band at ∼97 kDa present in the sample from the miR-16 target oligo beads and absent from the scrambled oligo beads.CC BY-NC-ND · PubMed Central
Fig. 6. Target mimic oligonucleotides specifically deplete their complementary miRNAs. (A and B) Small-RNA sequencing of plasma supernatant following incubation with scrambled oligonucleotide (x-axis) or miR-16 (n = 3; A) or let-7a (n = 2; B) target mimic (y-axis). (C and D) Deseq2 statistical analysis conducted with FDR threshold at 5% (adjusted P value ≤ 0.05) and fold change set to >2 shows the miRNAs that are depleted in a statistically significant manner by miR-16 target mimic (C) or let-7a target mimic (D). The UMI counts are normalized to the spike-in cel-miR-39.
Fig. 6. Target mimic oligonucleotides specifically deplete their complementary miRNAs. (A and B) Small-RNA sequencing of plasma supernatant following incubation with scrambled oligonucleotide (x-axis) or miR-16 (n = 3; A) or let-7a (n = 2; B) target mimic (y-axis). (C and D) Deseq2 statistical analysis conducted with FDR threshold at 5% (adjusted P value ≤ 0.05) and fold change set to >2 shows the miRNAs that are depleted in a statistically significant manner by miR-16 target mimic (C) or let-7a target mimic (D). The UMI counts are normalized to the spike-in cel-miR-39.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Geekiyanage, H., Rayatpisheh, S., Wohlschlegel, J. A., Brown, R., & Ambros, V.
Affiliations
  • Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605;
  • Department of Biological Chemistry, Geffen School of Medicine, University of California, Los Angeles, CA 90095;
  • Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2020-09-14
Volume
117
Issue
39
Pages
24213-24223
ISSN
  • 0027-8424
  • 1091-6490
DOI
10.1073/pnas.2008323117
PMC
PMC7533700
Cited by
148
References
64
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)
Funders
HHS | NIH | National Institute of General Medical Sciences (R01GM034028, R01GM088365, GM089778)

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