E3 Ligase RNF126 Directly Ubiquitinates Frataxin, Promoting Its Degradation: Identification of a Potential Therapeutic Target for Friedreich Ataxia

Abstract

Friedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. To date, there is no therapy to treat this condition. The amount of residual frataxin critically affects the severity of the disease; thus, attempts to restore physiological frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the frataxin E3 ligase may represent a strategy to achieve an increase in frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates frataxin ubiquitination and targets it for degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.

Keywords: E3 ligase; Friedreich ataxia; RNF126; frataxin; protein degradation; therapeutic target; ubiquitin.

Graphical abstract

Figure 1

Schematic Representation of the siRNA Screening Assay and Workflow (A) Schematic representation of the frataxin-ProLabel fusion construct used in the screening and the enzyme fragment complementation assay. The 6 kDa ProLabel tag encodes the inactive α fragment of the β-galactosidase enzyme, and it is expressed as a C-terminal tag fused to frataxin. The enzyme acceptor (EA) represents the complementary part of the enzyme. When the EA is added to the cells, together with the substrate, the two subunits combine to form a complete, active β-galactosidase enzyme that cleaves the chemiluminescent substrate, generating a luminescent signal proportional to the amount of frataxin-ProLabel. (B) Schematic representation of the screening workflow.

Figure 2

Silencing of RNF126 Promotes Accumulation of Frataxin Precursor and Mature Forms by Preventing Its Degradation (A) 293 Flp-In cell line stably expressing frataxin^1–210 was transfected with the indicated siRNA pools. Cell extracts were collected 48 or 72 hr post-transfection and analyzed by WB with anti-frataxin, anti-RNF126, and anti-tubulin antibody. Pre, precursor; int, intermediate; mat, mature frataxin. (B and C) Graphs representing relative frataxin precursor (B) or mature frataxin (C) abundance as quantitated by densitometric analysis of the blots in (A) and normalized with tubulin levels. (D) 293 Flp-In cell line stably expressing frataxin^1–210 was transfected with the indicated siRNA pools. Then, 48 hr after transfection, cells were treated with 100 nM actinomycin D for the indicated times. Cell extracts were analyzed by western blot with anti-frataxin, anti-RNF126, and anti-tubulin antibody. (E) Graph representing relative frataxin precursor abundance as quantitated by densitometric analysis of the blot in (D) and normalized with tubulin levels. Data represent the mean ± 1 SEM from four independent experiments. The p values were calculated with Student’s t test and were statistically significant (^∗∗p < 0.01) compared to non-targeting siRNA-transfected cells.

Figure 3

Expression of RNF126, But Not Its Catalytically Inactive Mutant, Promotes Frataxin Ubiquitination (A) HEK293 cells were transiently transfected with frataxin^1–210, HA-tagged ubiquitin (HA-Ub), and a control empty vector, RNF126, or its catalytically inactive mutant. Protein extracts were collected 40 hr post-transfection. Total cell extracts were analyzed by WB with anti-frataxin antibody or anti-tubulin as a loading control. Slower-migrating bands can be detected above the frataxin precursor, corresponding to mono- and polyubiquitinated frataxin. (B) Cells were transfected as in (A). Mono- and polyubiquitin-conjugated forms of frataxin can be detected by WB with anti-frataxin antibody on anti-HA immunoprecipitates. Control samples (excluded from these panels) were treated with 10 μM of the proteasome inhibitor MG132 as a positive control for the detection of ubiquitinated frataxin. ^∗Antibody heavy chain; ^∗∗antibody light chain.

Figure 4

Frataxin Precursor Interacts with RNF126 HEK293 cells were transiently transfected with the indicated constructs. Protein extracts were collected 40 hr post-transfection. (A) Proteins were immunoprecipitated with anti-frataxin (anti-fxn) antibody. Arrow indicates RNF126 coprecipitated with frataxin. ^∗Antibody heavy chain. (B) Proteins were immunoprecipitated with anti-RNF126 antibody. Upper left panel: short exposure. Upper right panel: long exposure. ^∗Non-specific band. Immunoprecipitate (IP) and total cell extract (tot) were probed on a WB with anti-frataxin (anti-fxn) antibody or anti-RNF126 antibody, as indicated. Pre, precursor int, intermediate; mat, mature frataxin; Ub-fxn, ubiquitinated frataxin; ex RNF126, overexpressed exogenous RNF126; end RNF126, endogenous RNF126.

Figure 5

In Vitro Ubiquitination Assay (A) In vitro ubiquitination assay was carried out with purified recombinant E1, UbcH5b as the E2 ubiquitin-conjugating enzyme, and different doses of GST-RNF126 as the E3 ubiquitin ligase, together with Ub, ATP, and recombinant frataxin precursor as a substrate. The reaction mixture was incubated for 60 min at 30°C. Reaction was stopped by the addition of 4× sample buffer. Proteins were separated on SDS-PAGE and analyzed by a WB with anti-frataxin antibody. (B) Ubiquitination assay was carried out with the indicated E2 ubiquitin-conjugating enzymes and 200 nM RNF126. Proteins were analyzed as in (A). (C) Ubiquitination assay was carried out as in (B), in the presence of the indicated concentration of 1,10-phenanthroline. Proteins were analyzed as in (A). Frataxin precursor, monoubiquitinated frataxin, and polyubiquitinated frataxin are indicated by arrows. Fxn precursor s.e. indicates a short exposure of the blot to appreciate precursor levels. ^∗Non-specific bands.

Figure 6

Silencing of RNF126 E3 Ligase Promotes Frataxin Accumulation in Cells Derived from FRDA Patients (A) Fibroblasts derived from two FRDA patients (FA816 and FA078) were transfected with the indicated siRNA. Cell extracts were collected 48 hr post-transfection and analyzed by WB with anti-frataxin antibody, anti-RNF126, and anti-tubulin. Tub, tubulin; mat fxn, mature frataxin. (B) Graph representing relative mature frataxin abundance as quantitated by densitometric analysis of the blots in (A) and normalized with tubulin levels. Data represent the mean ± 1 SEM from four independent experiments. The p values were calculated with Student’s t test and were statistically significant (^∗p < 0.05; ^∗∗p < 0.01) compared to non-targeting siRNA-transfected cells.