1. Cytosolic Iron-Sulfur Assembly Is Evolutionarily Tuned by a Cancer-Amplified Ubiquitin Ligase 
Jenny L. Weon, Seung Wook Yang, Patrick Ryan Potts 
Molecular Cell 
Volume 69, Issue 1, Pages e6 (January 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 113-125.e6DOI: (10.1016/j.molcel.2017.11.010)
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3. Figure 1
MAGE-F1 Binds the E3 Ligase NSE1 but Does Not Incorporate into the SMC5/6 Complex
(A) Immunoprecipitation (IP) of HA-NSE1 shows it binds MYC-MAGE-F1, similar to MYC-MAGE-G1, in HeLa cells.
(B) Endogenous NSE1 binds to MYC-MAGE-F1. HeLa cells were transfected with MYC-vector or MYC-MAGE-F1 constructs for 48 hr before immunoprecipitation with anti-MYC followed by SDS-PAGE and immunoblotting for endogenous NSE1.
(C) Pull-down of recombinant GST-NSE1 demonstrates binding to MYC-MAGE-F1, similar to MYC-MAGE-G1, but not the MYC-MAGE-F1 L87-88A dileucine mutant in vitro.
(D) Pull-down of MYC-SMC5 or MYC-SMC6 from HeLa cells demonstrates that FLAG-MAGE-F1 does not have the robust affinity for SMC5 or SMC6 as does FLAG-MAGE-G1.
(E) Pull-down of FLAG-MAGE-F1 or FLAG-MAGE-G1 in HeLa cells shows that endogenous SMC5 and SMC6 bind to MAGE-G1, but not MAGE-F1.
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4. Figure 2 MAGE-F1-NSE1 Targets MMS19 for Ubiquitination and Degradation
(A) HA-MAGE-F1, but not HA-MAGE-G1, robustly increases ubiquitination of MMS19. HeLa cells were transfected with the indicated constructs for 48 hr and treated with 10 μM MG132 for 4 hr followed by anti-MYC IP to pull-down ubiquitinated proteins followed by SDS-PAGE and immunoblotting for FLAG-MMS19.
(B) Mutation of five lysines in the C terminus of MMS19 (K993, K1002, K1007, K1008, and K1013; 5KR) disrupts MAGE-F1-induced ubiquitination. Cells were treated and lysates were prepared as noted in (A).
(C) Wild-type MAGE-F1, but not NSE1 binding defective mutant MAGE-F1 L87-88A (LA), induces ubiquitination of MMS19. Cells were treated and lysates were prepared as noted in (A).
(D) MAGE-F1-induced ubiquitination of MMS19 depends on the NSE1 ligase. Cells were transfected with the indicated siRNAs (siControl or siNSE1) and plasmids for 72 hr and treated with 10 μM MG132 for 4 hr followed by anti-MYC IP to pull-down ubiquitinated proteins followed by SDS-PAGE and immunoblotting for MMS19.
(E) MYC-MMS19 binds bacterially purified recombinant His-MAGE-F1-NSE1 complex, but not His-NSE1 alone, in vitro.
(F) Knockdown of endogenous MAGE-F1 or NSE1 in HeLa cells with two different siRNAs increases MMS19 protein levels.
(G) qRT-PCR analysis of MMS19 mRNA levels normalized to 18S rRNA in HeLa cells transfected with siMAGE-F1 or siNSE1 siRNAs. ns indicates p > Data are mean ± SD.
(H) Expression of MYC-MAGE-F1 wild-type (WT), but not L87-88A (LA) mutant, in HEK293 cells decreases MMS19 protein levels in a proteasome-dependent manner. Cells were treated with 10 μM MG132 (or DMSO control) for 4 hr, 72 hr after transfection.
(I) Knockout of MAGE-F1 increases MMS19 protein stability in HeLa cells. MAGE-F1 wild-type or knockout HeLa cells were treated with 100 μg/mL cycloheximide for the indicated times. Cell lysates were prepared, separated by SDS-PAGE, and immunoblotted for the indicated proteins.
(J) Denaturing His-ubiquitin pull-down from HeLa with ubiquitin mutants shows robust reduction of MMS19 ubiquitination in the presence of MAGE-F1 with the K48R ubiquitin mutant. Cells were treated with 10 μM MG132 for 5 hr, 48 hr after transfection.
See also Figure S1.
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5. Figure 3 MAGE-F1 Decreases Iron Incorporation into MMS19 Targets
(A) Immunoprecipitation of endogenous MMS19 target proteins after 55Fe treatment in HeLa cells demonstrates decreased incorporation of 55Fe either with MAGE-F1 overexpression or MMS19 knockdown, but not a non-MMS19 target protein (PPAT). Immunoblots below show MAGE-F1 transgene levels and MMS19 knockdown efficiency.
(B) Knockdown of NSE1 in HeLa cells abrogates MAGE-F1-mediated decrease of 55Fe incorporation into a target of MMS19. Immunoblots below show MAGE-F1 transgene levels and NSE1 knockdown efficiency.
(C) MAGE-F1 dileucine mutant is incapable of altering 55Fe incorporation into MMS19 target proteins in HeLa cells. Immunoblots below show MAGE-F1 transgene levels.
(D) MAGE-F1 effects on 55Fe incorporation into MMS19 targets are dependent on degradation of MMS19. HeLa cells were transfected with MAGE-F1 alone or in combination with wild-type MMS19 or non-ubiquitinatable MMS19 5KR. Incorporation of 55Fe into FANCJ and XPD was determined by immunoprecipitation and scintillation counting. Immunoblots below show expression of HA-MAGE-F1 and MMS19 transgenes.
(E) HeLa-Cas9 MAGE-F1 knockout cells exhibit greater 55Fe incorporation into MMS19-dependent proteins than their wild-type counterparts.
(F and G) MYC-MAGE-F1 expression in HEK293 cells causes decreased protein levels of MMS19 and downstream target proteins. Representative immunoblots shown in (F) and quantitation is shown in (G).
(H) Overexpression of MMS19 in the context of MAGE-F1 rescues FANCJ protein levels in HEK293 cells.
Data are mean ± SD. Asterisks indicate p < ns indicates p > See also Figure S2.
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6. Figure 4
MAGE-F1 Inhibits Homologous Recombination and Sensitizes Cells to DNA-Damaging Agents
(A) Increasing MAGE-F1 expression levels in HEK293 cells decreases rates of homologous recombination in a dose-dependent manner. Immunoblots below indicate MAGE-F1 transgene expression levels.
(B) The dileucine mutant of MAGE-F1 is incapable of inhibiting homologous recombination in HEK293 cells. Immunoblots below indicate MAGE-F1 transgene expression levels.
(C) Knockdown of MMS19 decreases rates of homologous recombination in a dose-dependent manner in HEK293 cells. Immunoblots below indicate MMS19 knockdown efficiency.
(D) MAGE-F1 effects on homologous recombination are dependent on ubiquitination of MMS19. HEK293 cells were transfected with MAGE-F1 alone or in combination with wild-type MMS19 or non-ubiquitinatable MMS19 5KR for 72 hr before homologous recombination rates were measured. Immunoblots below show levels of MMS19 and transgene-expressed MAGE-F1.
(E) Knockdown of downstream MMS19 targets shows that FANCJ and POLD1 phenocopy and likely mediate the defect in homologous recombination seen with MMS19 knockdown in HEK293 cells. Immunoblots shown to the right denote knockdown efficiency of siRNA targets using pooled siRNAs.
(F) MYC-MAGE-F1-expressing HeLa-Cas9 cells exhibit increased sensitivity to DNA damage by MMS compared to MYC-vector cells. Viability was normalized to untreated samples for both cell lines. Immunoblots below indicated levels of MAGE-F1 transgene expression.
(G) HeLa-Cas9 MAGE-F1 knockout cells are less sensitive than HeLa-Cas9 WT cells to MMS. Viability was normalized relative to WT cells.
(H) HeLa-Cas9 MAGE-F1 knockout cells are less sensitive than HeLa-Cas9 WT cells to UV. Viability was normalized relative to WT cells.
Data are mean ± SD. Asterisks indicate p < ns indicates p > See also Figure S3.
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7. Figure 5
Adaptive Pseudogenization of MAGE-F1 in Specific Mammalian Lineages
(A) Mammalian phylogenic tree indicating species with protein-coding or pseudogene (red) MAGE-F1. Note multiple gains and/or losses of MAGE-F1. Right: schematic of MAGE-F1 genes indicating sites of insertions, deletions, or mutations leading to in-frame stops is shown (asterisks).
(B) Sequences of those genetic alterations denoted with black asterisks in (A). Human, cat, and cow protein-coding MAGE-F1 sequences are shown for comparison.
(C) dN/dS ratio indicates those MAGE-F1 pseudogenes (24 species) are drifting toward neutral selection compared to protein-coding MAGE-F1 genes (36 species). See also Figure S4.
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8. Figure 6
MAGE-F1 Is Amplified in Multiple Cancers and Is Associated with Increased Tumor Mutational Burden
(A) MAGE-F1 is highly amplified in multiple cancer types.
(B) Amplification of MAGE-F1 is associated with increased MAGE-F1 mRNA levels in multiple tumor types.
(C) Significantly greater copy numbers of MAGE-F1 are observed in MAGE-F1-amplified lung squamous cell carcinoma cases.
(D) Total mutation burden is higher in MAGE-F1-amplified lung squamous cell carcinomas than in cases without amplification.
(E) A variety of mutational spectra are observed at higher frequencies in MAGE-F1-amplified lung squamous cell carcinomas.
(F) Significantly greater copy numbers of MAGE-F1 are observed in MAGE-F1-amplified head and neck squamous cell carcinoma cases.
(G) Total mutation burden is higher in MAGE-F1-amplified head and neck squamous cell carcinomas than in cases without amplification.
(H) Head and neck squamous cell carcinomas with high expression of both MAGE-F1 and NSE1 exhibit significantly worse clinical prognosis than those with low expression of both genes.
(I and J) Knockdown of MAGE-F1 in H520 (I) and HCC95 (J) lung squamous cell carcinoma cell lines with copy-number-amplified MAGE-F1 decreases xenograft tumor growth in mice (n = 6 per group).
(K) Stable expression of MAGE-F1 in H2170 lung squamous cell carcinoma cells without natural MAGE-F1 copy number amplification increases xenograft tumor growth rates in mice (n = 6 per group).
Data are mean ± SD. Asterisks indicate p < See also Figure S5.
Molecular Cell  , e6DOI: ( /j.molcel )
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