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. 2023 Dec 22;13(1):22952.
doi: 10.1038/s41598-023-50336-5.

TRIM25 dictates selective miRNA loading into extracellular vesicles during inflammation

Kayla E King  1   2 , Priyanka Ghosh  1   2 , Ann L Wozniak  3   4
Affiliations

TRIM25 dictates selective miRNA loading into extracellular vesicles during inflammation

Kayla E King et al. Sci Rep. .

Abstract

Extracellular vesicles (EVs) such as exosomes are loaded with specific biomolecules in order to perform cell-to-cell communication. Understanding the mechanism of selective cargo loading is important to better understand the physiological and pathological function of EVs. Here we describe a novel target of the E3 ligase TRIM25 and show that inflammation-mediated EV loading of the RNA binding protein FMR1 and its associated microRNA, miR-155, is promoted by TRIM25-mediated K63-ubiquitination of FMR1. This ubiquitination promotes an interaction between FMR1 and the EV loading machinery via the cleavage of the trafficking adaptor protein RILP. These interactions are lost when TRIM25 is knocked down. Loss of TRIM25 also prevents the loading of both FMR1 and miR-155. These findings suggest that inflammation-mediated loading of FMR1 and its associated microRNAs into the EV are dependent on K63-ubiquitination by TRIM25 and provide novel insights and tools to manipulate EV biogenesis for therapeutic benefit.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
TRIM25 Mediates FMR1 K63-ubiquitination. (A) Left panel shows a western blot analysis of HA-FMR1 immunoprecipitated from HeLa cells revealing that FMR1 is K63-Ub, particularly during inflammatory conditions. The right panel confirms FMR1 ubiquitination via a reversal IP. (B) Expression of cleaved RILP (cRILP) alone promotes the K63-Ub of FMR1 in HeLa. (C) Venn diagram of mass spectrometry analysis from proteins that immunoprecipitated with cRILP or ncRILP as well as EVs isolated from control, untreated THP-1 cells or inflammatory EVs isolated from THP-1 cells treated with LPS/ATP. (D) Cross-referencing the proteins that directly associate with cRILP and are loaded into inflammatory EVs yields a list of 10 proteins. One protein, TRIM25, is an E3 ligase. (E) In THP-1 cells, both TRIM25 and FMR1 are enriched it the EVs induced by inflammasome activation. (F) shRNA knockdown of TRIM25 in THP-1 cells blocks the K63 ubiquitination of FMR1. n = 3–5.
Figure 2
Figure 2
K63-Ubiquitinated FMR1 undergoes ESCRT-dependent EV loading. (A) Pulldown of FMR1 shows that it interacts with Hrs after inflammasome activation. This interaction is lost when TRIM25 is silenced (shT25). (B-C) Knockdown of TRIM25 does not alter the number of EVs secreted or the contents of known exosome markers. EV numbers were quantified via Bradford dye as performed by us previously. (D) Silencing TRIM25 dramatically reduces the EV enrichment of FMR1 during inflammation. (E) Correlation between the amount of intracellular TRIM25 and the amount of EV-associated FMR1. The amount of intracellular TRIM25 is positively correlated with EV FMR1. THP-1 cells were used for this figure. Data are shown as mean ± SEM; *p ≤ 0.05 for n = 3–5.
Figure 3
Figure 3
FMR Ubiquitination is Required for FMR1-mediated miRNA loading. (A) Total EV miRNA is not affected by TRIM25 knockdown in THP-1 cells. (B) Using THP-1 cells, miR-155 is enriched in inflammatory EVs. This effect is blocked when TRIM25 is silenced. (C) Analysis of FMR1-bound miR-155. FMR1 purified from both wild-type and sgTRIM25 CRISPR HeLa cells binds miR-155. FMR1 did not bind miR-155(mutant), a mutant engineered to eliminate the FMR1 binding motif. (D) FMR1 is K63Ub in HMC3 cells. (E) miR-155-5p is transferred and functional in a target cell. Conditioned media from an LPS/ATP-treated HMC3 macrophage reduces luciferase activity in a target expressing pmiRGlo-based luciferase reporter plasmid containing two miR-155-5p binding sites indicating the transfer of a functional miR-155-5p. Less functional miR-155-5p is transferred when target cells are treated with conditioned media from LPS/ATP-treated TRIM25 knockdown cells. (F) Using THP-1 cells, TRIM25 influences the EV loading of several miRNAs. Data are shown as mean ± SEM; *p ≤ 0.05 for n = 3–5.
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