The regulation of miR-155 strand selection by CELF2, FUBP1 and KSRP proteins

Abstract

The microRNA-155 exists in two forms, miR-155-5p and miR-155-3p, produced from either strand of the double-stranded precursor of miR-155 in a mutually exclusive manner. The more abundant and better-studied miR-155-5p has been implicated in numerous biological processes, with dysregulated expression observed in various human diseases. miR-155-5p plays an essential role in supporting inflammatory responses in macrophages. Activating macrophages with lipopolysaccharide (LPS) elevates miR-155-5p, while the anti-inflammatory cytokine interleukin-10 (IL10) reduces miR-155-5p levels. Recently, researchers have suggested that miR-155-3p also plays a role in macrophage function, although its specific function in this context remains unclear. We found that LPS stimulation of macrophages results first in the elevation of miR-155-3p levels, followed by an increase in miR-155-5p levels. In this paper, we investigate the mechanisms underlying the maturation of pre-miR-155 into either miR-155-5p or miR-155-3p. We describe the contribution of three RNA-binding proteins, CELF2, FUBP1 and KSRP, to pre-miR-155 processing. Our data suggest that CELF2 regulates the selection of miR-155-5p and miR-155-3p strands. FUBP1 may support the expression of miR-155-3p for specific subcellular functions, while KSRP appears to inhibit both miR-155-5p and miR-155-3p maturation without altering the relative expression of each strand.

Fig. 1

Increased interaction of FUBP1 protein with pre-miR-155 in response to LPS stimulation. RAW264.7 cells were transfected with biotinylated pre-miR-155 oligonucleotide and stimulated with LPS ± IL10 for 1 h before collecting the samples. (A) Expression levels of CELF2, FUBP1 and KSRP protein interacting with pre-miR-155 oligonucleotide were determined by immunoblotting. (B)The graphs show the CELF2, FUBP1, and KSRP band intensities in the pull-down sample, normalized to the same protein in the total cell lysate. One-way ANOVA with Tukey’s correction calculated the significance of the difference between the stimulations. ** p < 0.01, * p < 0.05, ns = not significant. The data represent the results of 5 experiments.

Fig. 2

Generation of FUBP1 and KSRP knockdown cells by CRISRP-Cas9 mediated gene silencing. RAW264.7/Cas9 cells transduced with FUBP1 sgRNA or KSRP sgRNA were treated with 2 µg/mL of doxycycline for 48 h to induce knockdown of FUBP1/KSRP proteins. FUBP1 and KSRP protein expressions were determined by immunoblotting. Data plotted represents FUBP1 and KSRP protein band intensity normalized to GAPDH. One-way ANOVA with Tukey’s correction determined the comparison indicated with braces. **** p < 0.0001, *** p < 0.001, ns = not significant. The data are representative of 3–5 experiments.

Fig. 3

CELF2, FUBP1, and KSRP deficiency alters the expression of miR-155-5p and miR-155-3p in response to LPS ± IL10 stimulation. FUBP1 KD, CELF2 KD, KSRP KD or the control RAW264.7 cells were stimulated with 1 ng/mL LPS ± 1 ng/mL of IL10 for (A) 2 and 4 h before total RNA extraction. The expression levels of miR-155-5p and miR-155-3p were determined by qPCR and normalized to snoRNA202 levels. The data plotted represents the expression levels of miR-155-5p and miR-155-3p levels normalized to the 2-hour LPS-stimulated control RAW264.7 cells. (B) The data from panel A were replotted as time courses for each cell type. miR-155-5p and miR-155-3p levels were normalized to the unstimulated sample of control RAW264.7 cells. Two-way ANOVA with Tukey’s correction determined the comparisons indicated with braces between different time points or cell types as * and the significance between stimulations as †. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, †††† p < 0.0001, ††† p < 0.001, †† p < 0.01, † p < 0.05, ns = not significant. The data are representative of 4 experiments.

Fig. 4

Expression of miR-155 target genes in CELF2 KD, FUBP1 KD, KSRP KD, or control RAW264.7 cells. Cells were stimulated with 1 ng/mL of LPS for 4 hours before extraction of total cellular RNA. The expression levels of Arg2, Apbb2 and Myd88 were determined by qPCR and normalized to GAPDH levels. Two-way ANOVA with Tukey’s correction determined the comparisons indicated with braces. **** p< 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. The data represents three experiments.

Fig. 5

FUBP1 and KSRP deficiency alters the expression of TNFα in response to LPS ± IL10. FUBP1 and KSRP KD cells were stimulated with 1 ng/mL LPS ± indicated concentration of IL10 for 1 h before collecting the cell culture supernatant. The level of TNFα in the supernatants was determined by ELISA. Two-way ANOVA with Tukey’s correction determined the comparisons indicated with braces. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. The data are representative of 3–5 experiments.

Fig. 6

STAT3 and SHIP1 dependence of miR-155-5p and miR-155-3p expression at 4 h. miR-155-5p or miR-155-3p expression levels in STAT3 WT and KO, or SHIP1 WT and KO cells were determined by qPCR and normalized to snoRNA202 levels. (A) Data were plotted to represent the RNA expression normalized to the LPS-stimulated sample of the STAT3 WT. (B) The data in panel A was replotted with RNA expression normalized to each cell’s own LPS-stimulated sample. Two-way ANOVA with Tukey’s correction determined the comparison between the stimulations indicated with braces. **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns = not significant. The data are representative of 3 experiments.

Fig. 7

Kinetics of miR-155-5p and miR-155-3p expression in STAT3/SHIP1 WT and KO cells. The expression level in SHIP1 WT and KO cells of miR-155-5p or miR-155-3p was determined by qPCR and normalized to snoRNA202 levels. (A) Kinetics of miR-155-5p and miR-155-3p expression with RNA normalized to the WT 0-hour sample. (B) The data plotted represents the comparison of LPS-stimulated miR-155 levels at 4 h between different cell types. (C) The data plotted represents the ratio of miR-155-3p and miR-155-5p. Two-way ANOVA with Tukey’s correction determined the significance of the difference in values (if any) in the LPS ± IL10 stimulated sample simultaneously. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. The data are representative of 3 experiments.

Fig. 8

The KH3 domain of KSRP and FUBP1 are essential for interacting with pre-miR-155. We expressed and purified KSRP/FUBP1 wildtype (WT) or KSRP/FUBP1 KH3 GDDG mutant proteins as described in the Materials and Methods section. (A) Coomassie Blue gel staining was used to assess the purity of the purified protein. (B) BLI biosensors loaded with biotinylated pre-miR-155 were dipped in wells containing KSRP WT, KSRP KH3 GDDG mutant, FUBP1 WT, FUBP1 KH3 GDDG mutant proteins for 10 min, followed by a dissociation step in the assay buffer for 20 min. (C) Data plotted to represent the BLI response of indicated proteins with biotinylated pre-miR-155. Unpaired Student’s t-test determined the comparison between the WT and KH3 GDDG mutant. **** p < 0.0001. The data represent the results of 3–5 experiments.

Fig. 9

Schematic representation of FUBP1/KSRP/CELF2 regulation, homology, and interaction. (A) The sequence alignment of the KH3 domain of FUBP1 and KSRP protein. Sequences in black and red indicate matching and mismatching sequences, respectively. (B) Schematic diagram showing the predicted interaction site of FUBP1, CELF2 and KSRP to pre-miR-155. The bases of pre-miR-155 are gray; blue bases show the miR-155-5p sequence, and black bases indicate the miR-155-3p sequence.

Fig. 10

Schematic representation of miR-155 regulation. Schematic diagram representing the regulation of miR-155-5p and miR-155-3p expression in response to LPS and IL10. The proteins in blue font represent the protein required for the shown action, and the proteins in orange font represent proteins that suppress the shown action.

Fig. 11

Schematic representation of IL10R downstream signaling process and potential mechanism on a different phase of pro-inflammatory response. Schematic diagram of IL10R downstream signaling pathway affecting various stages of pro-inflammatory response.