Article Figures & Data
Figures
- Figure 1.
miR-9-3p was more stable than miR-9-5p in a hypothalamic cell line. A, Gel electrophoresis of 32P-labeled miR-9-5p and miR-9-3p showing bands at their correct size: 23 and 22 nucleotides (nT), respectively. B, Representative gel image of miR-9-5p and miR-9-3p degradation over time (minutes) following incubation in hypothalamic-derived neuronal cell (IVB) lysate for 0, 15, 60, 120, or 240 min. C, Scatterplot of normalized densitometry values analyzed from gel images and fit with an exponential decay function (black line = miR-9-5p; red line = miR-9-3p; N = 4/group). D, Normalized densitometry values at the 60-min time point for miR-9-5p and miR-9-3p. Data are represented as mean ± SEM (N = 4/group). E, Mean half-lives of miR-9-5p and miR-9-3p derived from best-fit exponential decay functions. Results are represented as mean ± SEM (N = 4/group), and horizontal line overlaying the bar graph indicates the median. Data were analyzed using a two-sample t test.
- Figure 2.
miR-9-3p was more stable than miR-9-5p following transcriptional inhibition. Hypothalamic-derived IVB cells were treated with the transcriptional inhibitor actinomycin D for 2 h. Cells were lysed at 0, 15, and 60 min following treatment, total RNA was isolated, and RT-qPCR was performed for miR-9-5p and miR-9-3p (N = 5/group). Results were analyzed using the ΔΔCt method and are represented as mean fold change ± SEM, and horizontal line overlaying the bar graph indicates the median. Data were analyzed by two-way ANOVA with time and miR construct as factors. A Tukey’s post hoc test was performed to determine statistically significant differences between group means.
- Figure 3.
miR-9-3p was more stable than miR-9-5p in a brain region-dependent manner. A, Representative gel image of 32P-labeled miR-9-5p and miR-9-3p following incubation with rat brain lysate (N = 3/brain region). B, Scatterplot of normalized densitometry values analyzed from gel images and fit with an exponential decay function showing brain region-specific degradation kinetics of miR-9-5p and miR-9-3p. C, Mean half-lives of miR-9-5p and miR-9-3p in various brain regions derived from best-fit exponential decay functions. Results are represented as mean ± SEM (N = 3/group), and horizontal line overlaying the bar graph indicates the median. Data were analyzed by two-way ANOVA with brain region and miR construct as factors. A Tukey’s post hoc test was performed to determine statistically significant differences between group means.
- Figure 4.
Rapid miR-9-5p degradation in rat brain (SON) tissue lysate was dependent on protein concentration of the lysate. A, Representative gel image of 32P-labeled miR-9-5p following incubation with 1:10, 1:100, and 1:1000 dilutions of rat brain (SON) lysate, lysis buffer alone, or following SON lysate treatment with proteinase K for 60 min at 60°C. B, Scatterplot of normalized densitometry values analyzed from gel images and fit with an exponential decay function showing concentration-dependent degradation kinetics of miR-9-5p (N = 4/group).
- Figure 5.
Degradation of miR-9-5p was slower in primary astrocytes compared with rat brain (SON) tissue lysate. A, Representative gel image of 32P-labeled miR-9-5p following incubation with rat brain tissue (SON) lysate or rat primary astrocytes for 0, 1, 15, 60, or 120 min (N = 3). B, Scatterplot of normalized densitometry values analyzed from gel images and fit with an exponential decay function. C, Mean half-lives of miR-9-5p were derived from best-fit exponential decay functions. Results are represented as mean ± SEM (N = 3/group), and horizontal line overlaying the bar graph indicates the median. Data were analyzed using a two-sample t test.
- Figure 6.
Nucleotide sequences at both the 5′ and 3′ end contribute to miR-9 stability. A, Schematic diagram showing the nucleotide compositions of the miR-9 constructs: 5P = full-length canonical miR-9-5p, 3P = full-length canonical miR-9-3p, 5P* = miR-9-5p with a UGA to AGU substitution at the 3′ end, 3P* = miR-9-3p with an AGU to UGA substitution at the 3′ end, 5p isomiR = miR-9-5p with a U deletion at the 5′ end. Red font indicates nucleotide variation from canonical sequences. B, Representative gel image showing the degradation kinetics of 32P-labeled miR-9-5p, -3p, 5p*, 3p*, and 5p isomiR following incubation in rat brain (SON) lysate for 0, 1, 15, 60, or 120 min. C, Scatterplot of normalized densitometry values analyzed from gel images and fit with an exponential decay function showing that both 5′ and 3′ end modifications affect stability (N = 3–4/group). D, Normalized densitometry values at the 1-min time point for the miR-9-5p, 5p*, and isomiR constructs. Results are represented as mean ± SEM (N = 3–4/group). Data were analyzed by one-way ANOVA with miR construct as the categorical factor. A Tukey’s post hoc test was performed to determine statistically significant differences between group means. E, Normalized densitometry values at the 15-min time point for miR-9-3p and -3p*. Results are represented as mean ± SEM (N = 3–4/group), and horizontal line overlaying the bar graph indicates the median. Data were analyzed using a two-sample t test.
- Figure 7.
miR-9-5p and miR-9-3p were associated with distinct proteins in the rat vHIPP. A, Various length nucleotides matching the sequence for miR-9-5p (23, 16, 12 nT) and miR-9-3p (22, 16, 12 nT) were modified with a biotin tag on the 5′ end and incubated with lysate from the rat vHIPP. B, Biotinylated miR constructs were purified using streptavidin-coated magnetic beads and resolved on a 10% SDS-PAGE gel followed by Coomassie G-250 staining. Red box indicates gel bands that were dissected for in-gel digestion followed by mass spectrometry. C, Venn diagrams with number of unique proteins identified by mass spectrometry compared between miR-9-5p and miR-9-3p at various sequence lengths plus beads only, or D, comparison of identified proteins within miR-9-5p (23, 16, 12 nT) and miR-9-3p (22, 16, 12 nT). Red color numbers indicate proteins unique to each construct and black numbers indicate proteins common between constructs. Blue lettering indicates known RNA binding proteins.
Tables
Dataset Data structure Type of test Power Fig. 1D Normal distribution Two-sample t test p = 0.048 Fig. 1E Normal distribution Two-sample t test p = 0.063 Fig. 2 Normal distribution Two-way ANOVA Tukey’s HSD test Time: F(1,24) = 0.646, p = 0.533 miR: F(1,24) = 6.724, p = 0.016 Time × miR interaction: F(1,24) = 2.414, p = 0.111 Fig. 3C Normal distribution Two-way ANOVA Tukey’s HSD test Brain region: F(1,12) = 5.543, p = 0.020 miR: F(1,12) = 100.365, p = 3.511 × 10-7 Brain region × miR interaction: F(1,12) = 4.979, p = 0.027 Fig. 5C Normal distribution Two-sample t test p = 1.658 × 10–4 Fig. 6D Normal distribution One-way ANOVA F(2,9) = 10.119, p = 0.005 Fig. 6E Normal distribution Two-sample t test p = 0.045 - Table 2
List of unique proteins that were associated with various length nucleotides matching the sequence for miR-9-5p (23, 16, 12 nT) and miR-9-3p (22, 16, 12 nT), as identified by mass spectrometry using PEAKS software
5P 23 nT Protein D-3-phosphoglycerate dehydrogenase Glucose-6-phosphate 1-dehydrogenase Heterogeneous nuclear ribonucleoprotein 60-kDa heat shock protein Eukaryotic translation initiation factor 5 Cytoplasmic dynein 1 light intermediate chain 2 Tubulin α-1C chain Dihydrolipoyl dehydrogenase Pleiotropic regulator 1 5P 16 nT Protein Glyceraldehyde-3-phosphate dehydrogenase Aspartate–tRNA ligase cytoplasmic Mitochondrial-processing peptidase subunit α Kazrin Cytoplasmic dynein 1 light intermediate chain 2 26S proteasome regulatory subunit 6A Phosphatidylinositol 5-phosphate 4-kinase type-2 α Aldehyde dehydrogenase X 5P 12 nT Protein S-adenosylhomocysteine hydrolase-like protein 1 Pre-mRNA-processing factor 19 Keratin Type II cytoskeletal 73 Actin γ-enteric smooth muscle 3P 22 nT Protein Peripherin Excitatory amino acid transporter 1 Microtubule-associated protein 1A Tubulin α-1A chain Caveolae-associated protein 1 Vesicular glutamate transporter 1 26S proteasome regulatory subunit 6A 3P 16 nT Protein Caveolae-associated protein 1 Keratin Type II cytoskeletal 6A Plasminogen activator inhibitor 1 RNA-binding protein Sorting and assembly machinery component 50 homolog Keratin Type II cytoskeletal 2 epidermal Histone-binding protein RBBP7 3P 12 nT Protein Excitatory amino acid transporter 1 Pleiotropic regulator 1 Microtubule-associated protein 1A 26S proteasome regulatory subunit 6A Myelin proteolipid protein - Table 3
Table summarizing proteins identified by mass spectrometry that were associated with various length nucleotides matching the sequence for miR-9-5p (23, 16, 12 nT) and miR-9-3p (22, 16, 12 nT) and organized by functional class according to Panther gene ontology analysis
Cytoskeletal Membrane trafficking Protein modifying Transporter Metabolism Nucleic acid binding Translation miR-9-5p 22.2% – – – 33.3% 11.1% 11.1% 16 nT 12.5% – 25% – 37.5% – 12.5% 12 nT 40% – – – 20% – – miR-9-3p 28.6% 14.3% 14.3% 28.6% – – – 16 nT – 16.7% – – – 16.7% – 12 nT 20% – 20% 20% – – –
Extended Data Figure 1-1
AGO2 binds to 32P-labeled miR-9-5p and miR-9-3p. Representative gel image of 32P-labeled miR-9-5p, miR-9-3p, and an equimolar equivalent mixture of both following immunoprecipitation with (A) AGO2, (B) IgG control, and (C) AGO4 and β-actin. Download Figure 1-1, TIF file
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