PCBP2 recognizes the CELL motifs and has a functional role in intracellular retention of miRNA-155-3p.

A) Sequences of biotinylated oligos used as bait in pulldown experiment; CELL motifs (WT and mutated) are in grey, hEXO motifs (WT and mutated) are underlined. miRNA devoid of CELL motif (no-CELL), miRNA devoid of hEXO (no-hEXO).

B) Volcano plot comparing proteins bound to miRNA-155-3p no-CELL vs WT. Black curves represent the significant threshold at an a false-discovery rate (FDR) of 0.05 and S0 of 0.1. PCBP2 and SYNCRIP proteins are labeled in the plot.

C) CLIP of PCBP2 protein in murine hepatocytes. RT-qPCR analysis for miR-155-3p, miR-365-2-5p (CELL motif-devoid) and miR-31-3p (hEXO motif-devoid) is shown as IP/IgG. Data are the mean± SEM of three independent experiments.

D) RNA pull-down with the WT and mutated (no-CELL, no-hEXO) (sequences are reported in A) miR-155-3p followed by western blot for the indicated proteins (HSP90 is used as positive and GAPDH as negative controls respectively). Data are representative of three independent experiments.

E) RNA pull-down with the miR-365-2-5p followed by western blot for the indicated proteins. Data are representative of three independent experiments.

F) RNA pull-down by using the recombinant PCBP2 protein and with WT and mutated miR-155-3p (no-CELL) followed by western blot for PCBP2. Data are representative of three independent experiments.

G) CLIP of SYNCRIP protein in murine hepatocytes. RT-qPCR analysis for miR-155-3p is shown as IP/IgG. Data are the mean± SEM of three independent experiments.

H) (left and middle panels) EV miRNA-155-3p and miR-365-2-5p levels in shCTR and shPCBP2 cells analyzed by RT-qPCR. Data are expressed as ratio of miRNA expression in EVs with respect to the intracellular compartment (shCTR arbitrary value 1). Results are shown as the mean±S.E.M. of three independent experiments. (right panel) EV miRNA-155-3p levels in shCTR and shSYNCRIP cells analyzed by RT-qPCR. Data are expressed as ratio of miRNA expression in EVs with respect to the intracellular compartment (shCTR arbitrary value 1). Results are shown as the mean±S.E.M. of three independent experiments.

Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; **: p<0.01.

PCBP2 binding to miR-155-3p is SYNCRIP-dependent.

A) Co-immunoprecipitation of PCBP2 and SYNCRIP. Immunoprecipitations with rabbit polyclonal anti-PCBP2, mouse monoclonal anti-SYNCRIP and the relative preimmune IgG were performed on protein extracts from hepatocytes. GAPDH is used as negative control. Immunoblots representative of three independent experiments are shown.

B) Electrophoretic mobility shift assay (EMSA): interactions of miR-155-3p with the indicated protein extracts (shifts) and Abs (anti-SYNCRIP and anti-PCBP2) (supershift) are shown. Ultrashift shown in lane 5 demonstrates concurrent binding of SYNCRIP and PCBP2 to miR-155-3p.

C) Electrophoretic mobility shift assay (EMSA): interactions of miR-155-3p with protein extracts from shCTR (1), shPCBP2 (2) and shSYNCRIP (3) cells (shifts) and Abs (anti-SYNCRIP and anti-PCBP2) (supershift) are shown.

D) CLIP of PCBP2 protein in murine hepatocytes both WT (shCTR) and silenced for SYNCRIP (shSYNCRIP). RT-qPCR analysis for the expression of miR-155-3p is shown as IP/IgG. Data are the mean± SEM of three independent experiments.

PCBP2 binding to miR-155-3p is sequence dependent.

A) RNA pull-down with the WT and mutated (sequences are reported above) miR-26b-3p followed by western blot for the indicated proteins. Data are representative of three independent experiments.

B) RNA pull-down with the WT and mutated (sequences are reported above) miR-31-3p followed by western blot for the indicated proteins. Data are representative of three independent experiments. Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05.

A-B) CELL motifs (WT and mutated) are in grey, hEXO motifs (WT and mutated) are underlined.

PCBP2 functionally dominates on SYNCRIP EV-loading activity on a repertoire of miRNAs embedding CELL and hEXO motifs.

A) Volcano plot comparing miRNAs differently expressed from NGS data; miRNAs with Log2FC > 1 and Log2FC < −1 and p-value ≤ 0.10 were considered differently expressed. Downregulated miRNAs in shPCBP2 respect to shCTRL are represented as blue dots, upregulated miRNAs are represented as red dots.

B) Heatmap showing the Log2 fold enrichment (EV/CELL) of mature miRNAs in small extracellular vesicles derived from shCTRL cells versus shPCBP2 cells. miRNAs with Log2FE ≥ 1.0 and p-value ≤ 0.10 were considered to be differentially enriched.

C) List of selected miRNAs embedding CELL and/or hEXO motifs; consensus sequences are highlighted in grey or underlined respectively.

D) EV miRNA levels in shCTR and shPCBP2 cells analyzed by RT-qPCR. Data are expressed as ratio of miRNA expression in EVs with respect to the intracellular compartment (shCTR arbitrary value 1). Results are shown as the mean±S.E.M. of three independent experiments.

Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; **: p<0.01. E) EV miRNA levels in shCTR and shSYNCRIP cells analyzed by RT-qPCR. Data are expressed as ratio of miRNA expression in EVs with respect to the intracellular compartment (shCTR arbitrary value 1). Results are shown as the mean±S.E.M. of three independent experiments.

Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; **: p<0.01; ****: p<0.0001.

PCBP2 and SYNCRIP bind to several miRNAs embedding CELL and hEXO motif sequences.

A) CLIP of PCBP2 protein in murine hepatocytes. RT-qPCR analysis for the indicated miRNAs is shown as IP/IgG for each independent experiment (IgG arbitrary value 1). Data are the mean± SEM of three independent experiments. Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; **: p<0.01.

B) CLIP of SYNCRIP protein in murine hepatocytes. RT-qPCR analysis for the indicated miRNAs is shown as IP/IgG for each independent experiment (IgG arbitrary value 1). Data are the mean± SEM of three independent experiments.

Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; **: p<0.01; ***: p<0.001.

PCBP2 binding to miRNAs requires SYNCRIP.

CLIP of PCBP2 protein in murine hepatocytes both WT (shCTR) and silenced for SYNCRIP (shSYNCRIP). RT-qPCR analysis for the indicated miRNAs is shown as IP/IgG. Data are the mean± SEM of three independent experiments.

Data are considered statistically significant with p< 0.05 (Student’s T test). *: p<0.05; ***: p<0.001.

Schematic model of PCBP2/SYNCRIP dependent miRNAs compartmentalization.

Left) hEXO-SYNCRIP interaction promotes miRNAs secretion into EVs.

Right) SYNCRIP-dependent PCBP2-CELL motif interaction promotes miRNAs intracellular retention.

Biotinylated RNA oligonucleotides used in pull down experiments.

Primers for miRNA qPCR analysis.

Primers for gene expression qPCR analysis.

Oligos for shRNA cloning in pSUPER.retro.puro vector.

PCBP2 and SYNCRIP silencing.

a) Expression levels of PCBP2 in shCTR and shPCBP2 murine hepatocytes. Data are shown as the mean±S.E.M. of three independent experiments.

b) (Left panel) Western-blot analysis for PCBP2 on protein extracts from hepatocytes silenced for PCBP2 (3A shPCBP2) and relative control (3A shCTR). GAPDH has been used as loading control. The figure is representative of three independent experiments. (Right panel) Densitometric analysis of Western-blot signals. Data are shown as the mean±S.E.M. of three independent experiments.

c) Expression levels of SYNCRIP in shCTR and shSYNCRIP murine hepatocytes. Data are shown as the mean±S.E.M. of three independent experiments.

d) (Left panel) Western-blot analysis for SYNCRIP on protein extracts from hepatocytes silenced for SYNCRIP (3A shSYNCRIP) and relative control (3A shCTR). GAPDH has been used as loading control. The figure is representative of three independent experiments. (Right panel) Densitometric analysis of Western-blot signals. Data are shown as the mean±S.E.M. of three independent experiments.

shCTR, shPCBP2 and shSYNCRIP EV characterization.

a) Particle diameter (nm) and concentration (particles/ml) of EVs evaluated by Exoid (IZON) (Top: shCTR EVs, Middle: shPCBP2 EVs, Bottom: shSYNCRIP EVs).

d) Western-blot analysis for EV-specific (LAMP1, CD63, Synthenin) and intracellular (calnexin) markers on protein extracts from hepatocytes (WCE, whole cell extract) and hepatocyte-derived EVs (EVs).