A Strain: fl/fl ∆hep Strain: fl/fl ∆hep AnalysisAAV: ofLuc the ironLuc chaperonePCBP1-WT PCBP1PCBP1-∆Fe interactome:PCBP1-∆RNA PCBP1 variant intersection of DNA repair and iron traffickingAAV: Luc Luc WT ∆Fe ∆RNA PCBP1 −40

Lorena Novoa-Aponte a*, Sarju J. Patel a, Olga Protchenko a, James Wohlschlegel b, Caroline C. Philpott a −40 PCBP1, IHC PCBP1, GAPDH - a Genetics and Metabolism Section,a NIDDK, NIH, Bethesda, MD, USA. b Department of Biological Chemistry, UCLA, Los Angeles, CA, USA

C.C. Philpott, et al. *[email protected] 80 B P = 0.0262 60

40 1. Iron is essential but toxic 2. Increased DNA damage in cells lacking PCBP1 ns H&E ? + 20 C.C. Philpott, et al. Iron is used as an essential cofactor by several involved in DNA ⦿ Increased TUNEL in cells and tissue livers from mice lacking PCBP1. ? replication and repair. However, unchaperoned iron promotes redox ⦿ PCBP1 binds both, iron and single-stranded nucleicTG, nmol/mg 0 acids. stress that may affect DNA stability. Strain: Δhep Δhep Δhep ? ⦿ The iron binding activity of PCBP1 controls suppressionAAV: of DNA damage C WT ∆Fe ∆RNA PCBP1 variant ? Iron storage HEK293 cells Mouse Liver ? A 8 siNT+P1var 100 ? A 8 var

siPCBP1 2 ? Mammals use the iron siNT+P1 ns var Fe-S cluster assembly 6 siPCBP1+P1siPCBP1 80 ADI1 TUNEL chaperone PCBP1 to var ? 6 siPCBP1+P1 metalate several iron- 60 = 0.0468 = 0.0354 = 0.0465 cell / mm population P P P population + dependent enzymes + 4 + 4 ? 40 Degradation of HIF1α Fig. 3. Iron chaperone-mediated handling of cytosolic labile iron pool. Divalent metal transporter 1 (DMT1) and Zip14, located on plasma membrane or endocytic vesicles, imports non-transferrin-bound or transferrin-bound iron, 20 respectively. Iron enters the cytosolic labile iron pool (LIP) as Fe(II) and is largely coordinated by reduced GSH and bound to iron chaperones, PCBPs. PCBP1 and 2 2 TUNEL PCBP2 efficiently distribute iron for storage, non-heme iron cofactor assembly, or efflux through ferroportin (Fpn1). a, PCBP1 (and to a lesser extent, PCBP2) % TUNEL facilitates iron sequestration into ferritin,? which can be delivered to mitochondria by unknown mechanism for heme or FeeS cluster synthesis. b, PCBP1 form a % TUNEL 0 complex with cytosolic BolA2 via a bridging Fe-GS ligand. Iron provided by PCBP1-BolA2 complex can be combinedTUNEL with sulfur (S) compound, with the help of other / of polyproline motifs 0 cytosolic FeeS cluster assembly and facilitateADI1 the [2Fee2S] cluster formation on BolA2-Glrx3 distribution system. c, PCBP1 delivers iron to mononuclear Strain: fl/fl Δhep Δhep Δhep Δhep P1var: WT - WT ∆RNA - ∆RNA ∆Fe - ∆Fe iron prolyl hydroxylase (PHD2), which regulates the degradation of hypoxia inducible factor 1-α (HIF1α). d, PCBP1 also metallates the dinuclear iron 0 AAV: Luc Luc WT ∆Fe ∆RNA enzyme deoxyhypusine hydroxylase (DOHH), which catalyzes the hydroxylation of hypusine (Hpu) on eukaryotic translation initiation factor 5A (elF5A) to promote P1var: WT - WT ∆RNA - ∆RNA ∆Fe - ∆Fe translation of polyproline motifs. DAPI Philpott, CC, et al. BBA - Molecular Cell Research (2020) Sarju, P. et al. PNAS (2021) PCBP1 variant state [31]. For this reason, PCBP1 cannot be delivering iron directly to PCBP1. Again, PCBP1 demonstrated iron-dependent binding to DOHH B siNT siPCBP1 Fig.the 3. Iron enzymatic chaperone-mediated active site handling but instead of cytosolic is likely labile releasing iron pool. iron to the in cells by coimmunoprecipitation. Because ferritin and the non-heme siNT + P1var siPCBP1 + P1var Divalentcarboxylate metal transporter residues that 1 (DMT1) line the and pore Zip14, of 3-fold located symmetry on plasma that membrane leads to or endocyticiron enzymes vesicles, targeted imports non-transferrin-bound by PCBP1 are structurally or transferrin-bound dissimilar, we iron, have B 150 siNT siPCBP1 respectively.the interior. Iron entersThis mechanism the cytosolic of labile associative iron pool transfer (LIP) as makes Fe(II) and the is role largely of a coordinatedproposed by reduced that the GSH interactions and bound are to iron largely chaperones, mediated PCBPs. through PCBP1 bridging and D 400 ns PCBP2bridging efficiently iron distribute ligand more iron likely.for storage, non-heme iron cofactor assembly, or effluxiron through ligands ferroportin that may (Fpn1). include a, PCBP1 GSH. (and Additional to a lesser electrostatic extent, PCBP2) interac- var var facilitates iron sequestration into ferritin, which can be delivered to mitochondria by unknown mechanism for heme or FeeS cluster synthesis. b, PCBP1 form a siNT + P1 PCBP1 can deliver iron to non-heme enzymes with mono- and di- tions, for example, cannot be ruled out, but may not be primarily re- 150 siPCBP1 + P1 complexnuclear with iron cytosolic centers BolA2 (Fig.via a 3 bridgingc, d) [32 Fe-GS]. This ligand. activity Iron provided was initially by PCBP1-BolA2 ob- sponsible complex can for be complex combined formation. with sulfur (S) compound, with the help of other cytosolic FeeS cluster assembly proteins and facilitate the [2Fee2S] cluster formation on BolA2-Glrx3 distribution system. c, PCBP1 delivers iron to mononuclear 300 served in the prolyl hydroxylases (PHDs) that regulate the degradation 100 iron enzyme prolyl hydroxylase (PHD2), which regulates the degradation of hypoxia inducible factor 1-α (HIF1α). d, PCBP1 also metallates the dinuclear iron of hypoxia inducible factor 1-α in cells [33]. Cells from which PCBP1 is e enzyme deoxyhypusine hydroxylase (DOHH), which catalyzes the hydroxylation of hypusine5. Interaction (Hpu) on eukaryotic of PCBP1 translation with cytosolic initiation factor Fe S 5A assembly (elF5A) to machinery promote translationdepleted of polyprolineexhibit loss motifs. of the iron cofactors from PHD2 and concomitant through BolA2 = 0.0159 = 0.0255 loss of prolyl hydroxylase activity, reduction of hydroxylated HIF1-α, 200 = 0.0079 P P P and accumulation of active HIF1-α. Similarly, PCBP1-depleted cells A proteomics analysis of the PCBP1 interactome indicated that 100 state [31]. For this reason, PCBP1 cannot be delivering iron directly to PCBP1. Again, PCBP1 demonstrated iron-dependent binding to DOHH 50 ALT, U/L exhibit loss of activity of the related asparagyl hydroxylase and PCBP1 PCBP1 was capable of binding many different protein components in % Cell Viability the enzymatic active site but instead is likely releasing iron to the in cells by coimmunoprecipitation. Because ferritin and the non-heme was found to directly interact with both of these mononuclear iron the cell, including ferritins, other PCBPs, and some non-heme iron en- 100 carboxylate residues that line the pore of 3-fold symmetry that leads to iron enzymes targeted by PCBP1 are structurally dissimilar, we have enzymes. A recent publication also indicates that acireductone dioxy- zymes. Inspection of the interactome for possible co-chaperone, rather the interior. This mechanism of associative transfer makes the role of a proposed that the interactions are largely mediated through bridging genase 1, a mono-nuclear iron enzyme involved in the methionine than client, proteins revealed BolA2 as a candidate (Fig. 3b) [26]. bridging iron ligand more likely. iron ligands that may include GSH. Additional electrostatic interac- salvage pathway, is also metallated by PCBP1 in cells [34]. BolA2 homologues have been characterized in budding and fission 0 0 PCBP1 can deliver iron to non-heme enzymes with mono- and di- tions, for example, cannot be ruled out, but may not be primarily re- 50 Deoxyhypusine hydroxylase (DOHH) is a di-nuclear iron enzyme yeast as components of a cytosolic/nuclear [2Fee2S] carrier complex EV P1-WT P1-∆RNA P1-∆Fe nuclear iron centers (Fig. 3c, d) [32]. This activity was initially ob- sponsible for complex formation. Strain: % Cell Viability that is structurally unrelated to PHDs or ferritins, but also depends on that includes the cytosolic monothiol glutaredoxin, Grx3 [36–40]. In fl/fl Δhep Δhep Δhep Δhep served in the prolyl hydroxylases (PHDs) that regulate the degradation PCBP1 for efficient metalation of its peroxo-bridged, di-nuclear iron yeast this complex is proposed to transfer [2Fee2S] clusters to iron- PCBP1-Flag Variants of hypoxia inducible factor 1-α in cells [33]. Cells from which PCBP1 is e AAV: Luc Luc WT ∆Fe ∆RNA center [35]. Cells lacking PCBP1 or PCBP2, but not wild type HEK cells, 5. Interactionregulatory transcription of PCBP1 with factors, cytosolic which Fe directlyS assembly affect machinery their activity depleted exhibit loss of the iron cofactors from PHD2 and concomitant lost DOHH activity when exposed to low levels of iron chelators. Iron through[41–44 BolA2]. In mammalian cells, BolA2-Glrx3 complexes also function as loss of prolyl hydroxylase activity, reduction of hydroxylated HIF1-α, cofactors were selectively lost from DOHH under these conditions and [2Fe-2S] chaperones; the only known recipient of these clusters in Fig. 5 | RNA or Iron binding activityPCBP1 of PCBP1 variant are associated with different phenotypes in PCBP1 and accumulation of active HIF1-α. Similarly, PCBP1-depleted cells 0 could be restored in vitro with iron or with cell lysates containing Amammalian proteomics cells analysis is Ciapin of 1, the an PCBP1early-acting interactome component indicated of the cytosolic that depleted cells. (A) Iron binding activity of PCBP1 is required to suppress DNA damage in PCBP1- exhibit loss of activity of the related asparagyl hydroxylase and PCBP1 PCBP1 was capable of binding many different protein components in EV P1-WT P1-∆RNA P1-∆Fe was found to directly interact with both of these mononuclear iron depleted cells. Tetracycline-inducible stable cell lines expressing PCBP1-Flag variants of wild type (WT), the cell, including ferritins, other PCBPs, and some non-heme iron en- enzymes. A recent publication also indicates that acireductone dioxy- zymes. Inspection of the interactome for possible co-chaperone, rather RNA-binding mutant (∆RNA) or iron-binding mutant (∆Fe)PCBP1-Flag were transfected Variants with nontargeting (NT) or genase 1, a mono-nuclear iron enzyme involved in the methionine than client, proteins revealed BolA2Fig. as a candidate 6 | RNA (Fig. 3b) [-26].and iron-binding activities3’UTR- PCBP1of PCBP1 siRNA and treated are with associated (+P1var) or without (with-) doxycycline different to induce PCBP1 phenotypes variants in PCBP1 salvage pathway, is also metallated by PCBP1 in cells [34]. BolA2 homologues have been characterized in budding and fission (P1var) for 48 h. Cells were fixed and stained with TUNEL/PI. Bar graph shows the percentage of Deoxyhypusine hydroxylase (DOHH) is a di-nuclear iron enzyme yeast as components of a cytosolic/nucleardepleted [2Fee2S] carrier hepatocytes. complex (A) TransductionTUNEL+ of cells AAV in each constructs group. (B) RNA binding with activity PCBP1 of PCBP1 wild is required type for cell (WT), viability in RNA PCBP1--binding mutant that is structurally unrelated to PHDs or ferritins, but also depends on that includes the cytosolic monothiol glutaredoxin, Grx3 [36–40]. In Fig. 5 | RNAdepleted or Iron cells. binding Tetracycline activity-inducible of stablePCBP1 cell arelines associatedexpressing empty with vector different (EV) or PCBP1phenotypes-Flag in PCBP1 PCBP1 for efficient metalation of its peroxo-bridged, di-nuclear iron yeast this complex is proposed to transfer(∆RNA) [2Fee2S] clustersor iron to iron--binding mutant (∆Fe) in PCBP1 wild type (fl/fl) and deleted (∆hep) livers. Representative depleted cells.variants (A) (P1Ironvar) of bindingwild type activity(WT), RNA of- bindingPCBP1 mutant is required (∆RNA) orto ironsuppress-binding mutantDNA damage(∆Fe) were in PCBP1- center [35]. Cells lacking PCBP1 or PCBP2, but not wild type HEK cells, regulatory transcription factors, which directly affect their activity var lost DOHH activity when exposed to low levels of iron chelators. Iron [41–44]. In mammalian cells, BolA2-Glrx3immunohistochemistry complexes also function as showing depletedthe distribution cells.transfectedTetracycline with ofnontargeting PCBP1-inducible (NT) stable orin 3’UTR AAV cell-PCBP1 lines-transduced siRNAexpressing and treated PCBP1 liverswith -doxycyclineFlag ( variantsleft (+P1). AAV of) wildfor 72- typeluciferase (WT), (Luc) cofactors were selectively lost from DOHH under these conditions and [2Fe-2S] chaperones; the only known recipient of these clusters in RNA-bindingh as mutant indicated. (∆RNA) Cell viability or iron was- bindingassessed mutantusing CCK (∆Fe)-8 assays were in transfectedtriplicates. All datawith are nontargeting mean ± s.d. of (NT) or could be restored in vitro with iron or with cell lysates containing mammalian cells is Ciapin 1, an early-acting component of the cytosolic n = 3 independent experiments. fl/fl ∆hep used as negative control. Representative3’UTR-PCBP1 immunoblotsiRNA and treated of with PCBP1 (+P1var) or in without liver (- )lysates doxycycline from to induce PCBP1 PCBP1 variantsand PCBP1 mice injected with the indicated (P1AAVvar) -forconstructs. 48 h. Cells were GAPDH fixed and stained used with as TUNEL/PI. a loading Bar graphcontrol shows ( rightthe percentage). (B) Persistence of of TUNEL+ cells in each group. (B) RNA binding activity of PCBP1 is required for cell viability in fl/PCBP1fl - steatosis in livers transduced withdepleted PCBP1 cells. Tetracycline∆Fe. Hematoxylin-inducible stable & cell eosin lines expressing (H&E) emptystaining vector of(EV) PCBP1 or PCBP1-Flagand PCBP1 ∆hep mice injected with AAVs (leftvariants). Total (P1 varacylglycerols) of wild type (WT), (TG) RNA-binding in livers mutant of (∆RNA) PCBP1 or iron∆hep-binding mice mutant injected (∆Fe) were with the transfected with nontargeting (NT) or 3’UTR-PCBP1 siRNA and treated with doxycycline∆hep (+P1var) for 72 ∆hep PCBP1 variant AAVs (right). (C)hIncreased as indicated. Cell TUNEL viability -waspositive assessed cells using CCKin liver-8 assays from in triplicates. PCBP1 All data miceare mean and ± s.d. PCBP1of mice expressing ∆Fe mutant. Representativen = 3 independent experiments. images of liver sections stained with TUNEL and DAPI (left) with quantitation (right). (D) RNA-binding activity of PCBP1 is required to suppress hepatocyte cell death. Plasma ALT levels in PCBP1fl/fl and PCBP1 ∆hep mice transduced with AAVs. Data represent mean ± SD. P values calculated using one-way ANOVA and unpaired student’s t-test. See also Fig. S6. ImageImage ID: ID:0005275_01 0005273_02 PagePage 1 1 AcquireAcquire Time: Time: Jun Jun 8, 2021 7, 2021 11:32:36 5:40:39 AMPM

Analysis of theAcquisition iron Information chaperone PCBP1 interactome: Acquisition Information # Image ID Acquire Time ChannelsImageResolution ID: 0005277_01Intensities Quality Analysis Image Name Comment Page 1 # Image ID Acquire Time Channels Resolution Intensities Quality Analysis Image Name Comment intersection1 0005273_02 of JunDNA 7, 2021 5:40:39 PMrepairAcquire700 800 Time:169uma- γJunandH2AX Auto8, 2021 Auto iron 3:53:29lowest Manual PM trafficking0005273_02 gH2Ax_800_gel2 1 0005275_01 Jun 8, 2021 11:32:36 AM 700 800 169umImageAuto ID: Auto0005273_01lowest Manual 0005275_01 PCBP1_800_gel1 Page 1 WCE Acquirea-PCBP1Cyt Time: Jun 7, 2021 5:40:39Nuc PM Image Display Values WCE ImageCyt ID: 0005275_02 Nuc Page 1 Image DisplayChannel ValuesColor + Minimum Maximum K + Acquire Time: Jun 8, 2021 11:32:36 AM C AcquisitionChannel 800Color InformationGray Scale (Black on White)Minimum0.0193Maximum4.52 K 0 + + 3. DNA repair pathways upregulated# 800Image IDinGray PCBP1 ScaleAcquire (Black Timemutant on White) 5.25Channels110Resolution0 Intensities4. IncreasedQuality Analysis rateImage of NameDSBsC Comment on cells lacking PCBP1 Image ID: 0005276_01 Page 1 DDR SiPCBP1 NT

Acquisition Information SiBolA2 NT 1 0005277_01 Jun 8, 2021 3:53:29 PM 700 800 169um SiPCBP1 Auto Auto lowest Manual 0005277_01 Tubulin_700_gel1 SiPCBP1 NT SiBolA2

SiBolA2 Acquire Time: Jun 8, 2021 11:39:21 AM # Image ID Acquire Time Channels⦿ResolutionThe CHKIntensities1 sensorQuality(requiredAnalysis Imagefor NamecheckpointComment -mediated activation of DNA Transcriptomic analysis of PCBP1 depleted livers (RNA-Seq, KO vs WT) : DDR SiPCBP1 NT SiBolA2 SiPCBP1 NT SiPCBP1 NT SiBolA2 1 0005273_01 Jun 7, 2021 5:40:39 PMSiBolA2 700 800 169umdamageAutoresponse Auto lowest DDR),Manual and0005273_01the histonegH2Ax_800_gel2H2AX (early responder upon Acquisition Information a-Tubulin BER NER MMR DR FA ImageHDR DisplayNHEJ ValuesTLS NP other DNA double-strand break (DSB) occurrence) were upregulated. 4 # Image ID Acquire TimeWCE Channels ResolutionCyt Intensities Quality AnalysisNuc Image Name Comment 3 Channel ColorAcquisition Information Minimum MaximumImageK ID: 0005277_02 Page 1 1 0005275_02Image DisplayJun 8, 2021 Values 11:32:36 AM 700 800 169um⦿ DSBsAutotrigger Auto phosphorylationlowest Manual 0005275_02of H2AXPCBP1_800_gel1producing gH2AX. 2 700 Gray# ImageScale ID(BlackAcquire on White) Time 2.99 AcquireChannels81.9 Time:Resolution0 Jun 8,Intensities 2021 3:53:29Quality Analysis PM Image Name Comment

Channel Color Minimum Maximum K + 1 1 0005276_01+ Jun 8, 2021 11:39:21 AM 700 800⦿ Increased169um Autolevels Auto lowestof gHManual2AX suggest0005276_01thatLamin_800_gel2DSBs occur in cells lacking PCBP1. C 0 800 Gray Scale (Black on White) 0.0193 4.66 0

Log2 FC a-Lamin -1 Image Display Values

-255 Image Display Values WCE Cyt Nuc DDR NT 5 Channel Color Minimum Maximum K SiPCBP1 SiBolA2 NT SiPCBP1 - + NT SiPCBP1 SiBolA2 -3 SiBolA2 800 GrayChannel ScaleColor (Black on White) 5.25 Minimum110 Maximum0 K CDC25C CDC25C CDC25C Acquisition Information + PARPBP PARPBP PARPBP 800 Gray Scale+ (Black on White) 0.126 12.0 0 PCBP1 38 kDa 4 44 C ⦿ DSBs are the most deleterious DNA lesions. # Image ID Acquire Time Channels Resolution IntensitiesPCBP1 Quality Analysis Image Name Comment FANCD2 FANCD2 FANCD2 Image ID: 0005276_03 Page 1

1 0005277_02 Jun 8, 2021 3:53:29 PM 700 800 169um Auto Auto lowest Manual 0005277_02DDR Tubulin_700_gel1 PLK3 PLK3

33 PLK3 RAD51B 3 RAD51B RAD51B Acquire Time: Jun 8, 2021 11:39:21 AM NT SiPCBP1 SiBolA2 CHEK1 CHEK1 CHEK1 NT SiPCBP1 NT SiPCBP1 SiBolA2 SiBolA2 H2AX 15 kDa RAD51C RAD51C RAD51C RAD51C H2AX RAD51C RAD51C γ EME1 EME1 EME1 POLQ POLQ POLQ POLQ POLQ POLQ RAD51 RAD51 RAD51 RAD51 RAD51 2 22 RAD51 ⦿ Does PCBP1 play a role in the GADD45G GADD45G GADD45G Tubulin 50 kDa RRM2 RRM2 EID3 EID3 RRM2 EID3 EXO1 EXO1 EXO1 EXO1 FANCA FANCA EXO1 EXO1 BARD1 BARD1 FANCA RBBP8 RBBP8 BARD1 BARD1 BARD1 RBBP8 BARD1 POLE2 POLE2 POLE2 POLE2 POLE2 POLE2 Image ID: 0005276_02 maintenance ofPageenzymes 1 involved in TELO2 TELO2 CHAF1A

CHAF1A Tubulin TELO2 FEN1 FEN1 FEN1 FEN1

Image Display CHAF1A Values FEN1 FEN1 RAD54L RAD54L KAT5 KAT5 RAD54L KAT5 RMI2 RMI2 TTK RMI2 NEIL3 TTK PRPF19 RMI2 NEIL3 RMI2 PRPF19 H2AX H2AX POLE POLE TTK POLE NEIL3 POLE PRPF19 RMI2 H2AX POLE POLE 11 Acquire Time: Jun 8, 2021 11:39:21 AM 1 Channel ColorAcquisition Information Minimum Maximum K DDR that need iron for function? Log2 FC Log2 FC Log2 FC 700 Gray# ScaleImage ID(Black Acquireon White) Time 2.99 Channels81.9 Resolution0LaminIntensities A/C Quality Analysis Image Name Comment Lamin 70 kDa 0 00 1 0005276_03 Jun 8, 2021 11:39:21 AM 700 800 169um Auto Auto lowest Manual 0005276_03 Lamin_800_gel2 Fanconi anemia Homology-Directed Repair Cell cycle DSB -1 -1-1 Acquisition Information

Image Display Values CLK2 CLK2 # Image ID CLK2 Acquire Time Channels Resolution Intensities Quality Analysis Image Name Comment SMC5 SMC5 PER1 PER1 SMC5 PER1 MUS81 MUS81 from the Fanconi anemia, homologyMUS81 -directed DNA repair pathways, -2 -2-2 Channel1 0005276_02Color Jun 8, 2021 11:39:21 AMMinimum700 800Maximum169um K Auto Auto lowest Manual 0005276_02 Lamin_800_gel2 HUS1 HUS1 and cell cycle control were upregulated in PCBP1800 depletedGray ScaleHUS1 (Black livers on White) 0.126 12.0 0 RAD52 RAD52 RAD52 �H2Ax �H2Ax MUTYH MUTYH -3 MUTYH -3-3 Image Display Values Channel Color Minimum Maximum K 800 Gray Scale (Black on White) 0.126 12.0 0 Analysis of the iron chaperone PCBP1 interactome: intersection of DNA repair and iron trafficking

5. Several proteins involved in DDR use iron for function 6. Exploring PCBP1 interactome

Iron centers Fe-S clusters CoIP and proximity labeling approaches to identify PCBP1 interacting proteins

FANCJ DDX11 DNA repair

PUF60

DHX36 ALKBH1 EP400 TOP2A DNA SMARCA5 MSH6 POLRMT DNAJC2 RECQL CHD1 POLA1 RTEL1 PDS5B

NONO UBA1 RFC1 DDB1 DDX23 helicases POLR3B CHD3 DDX18 DDX56 SNRNP200 DNA SON CHD4 DHX30 BAZ1B BPTF DNMT1 ALKBH2 POLR2B ZC3HAV1 DHX16 DDX24 POLR1A TOP1 DDX10 APBB1 DHX8 demethylases NOL9 CHAF1B UHRF1 HELZ XPD DNA2 HAT1 DHX37 PDS5A DHX38

PRPF4B PRKDC NOC3L FUBP3 ING3 TRIR ZNF326 EIF4G1 DDX52 ARPC4 ALKBH3 HMGN2 DDX54 UQCRQ RBMS1 ZFR PUM3 RBM15 YTHDF1 STAG2 SRCAP GTPBP1 LARP4B RBM12 DDX20 TOP2B BAZ1A KIF22 LIG3 EIF4G2 SUZ12 NSRP1 CHAF1A H2AC1 RIF1 MOV10 EIF4G3 XRN2 RNASEH2B ACTR2 DKC1 POLA1 POLE RAB14 RCL1KRT9 RBM12B COPB2 FXR2 BMS1 LBR CYC1 KIFBP RRP1B XRN1 RBM26 KRT6B FAU TRMT10C PRRC2C TBL3 WDR3 HELLS RBM14 DNA MCM6 RRP8 UBTF SRSF11 Ribonucleotide POLDIP3 FUS HNRNPDL MCM3 LSG1 MEPCE ABCF2 SMC2 NOP56 RPS19 WDR36 ATXN2 RRM2 PUM2 NOL6 UTP6 MATR3 PRKACA SMC4 RPS16 KRT6A CEBPZ ADNPSLC25A11 YTHDC2 WDR46 YLPM1 CENPV MCM5 CNP CHTOP FXR1 SLC16A1 reductase SFXN1 TNPO1 PCBP1 SLK ZNF318 TAB2 UTP20 NEMF RPL36 LTV1 UBAP2 TWF1 UPF1 RPL17 SUGP2 POLD REV3L TNRC6B NOP14 GAR1GTF3C5 NAA16 WDR75 ETF1 NOC2L TPP2 MDN1 RPL14 PNO1 GTF2F1 RBM28 UTP15 TFCP2 NOL10 RBM4B LMF2 IDE CEP170 MYBBP1A NCOR1 UTP14A MOGS PES1 TSR1 KRT16 FTSJ3 NIFK GATAD2A KNL1 PCBP4 HBS1L MAP3K7 CAPZA2 SUGP1 CGN PHF2 NOP58 AKAP17A NOL8 SMC3 IDH3B SMC1A PYGL KRT10 GIGYF2 HADHA BRIX1 TEX10 TIMM44 DNA PSMD2 USP7 POGZ ZC3H11A KRT5 NNT MDC1 RPS14 WDR18 RRP12 GTF3C1 DIMT1 PRIM2 FKBP5 LARP7 RPF2 CELF2 NKRF LRRC47 PSMD6 SBDS DNAJC7 MRTO4 KRT2 ZNF622 MYH11 PSMD8 PSMD3 NXF1 Histone PSMC1 CDK12 PUM1 CKAP5 LRIF1 MRPL48 ATP5MG CDC5L WDR1 FAM98A PCBP3 MTA2 PHF8 PRRC2A KIFC1LRRC40 STAU2 SMG7 GCN1 PPP4C PTPN11 CNN3 PSMD12 EIF2AK2 ABRAXAS2 GNL2 MARK3 GLUD1 MBNL2 HMGB3 PBRM1 ILF3 demethylases SMARCA4 PSMD13 PPM1G ATG2B KRT77 NOP2 RANBP2 ANKRD17 MAPK1 NUP205 TARDBP SLC3A2 NUP85 KRT1 KPNA2 PPP1R10 EIF2B4 MTHFD1L CUL3 SMARCA2 KHDRBS1 CTNNA1 NCOA2 ADARSRBD1 PCBP2 FAM120A ALYREF CEP85 SLC25A12 CCAR1 TJP1 STRBP KDM1A SRPK1 YTHDF3 NUP107 NEDD1 CISD2 IQGAP2 TRIP12 CAD DEK AGPS ATP2B1 YWHAH LAS1L POR VARS1 POLA1 ENO2 ELAVL1 PHIP FERMT2 MBNL1 SERPINB6 CKAP4 PLRG1 KIF2C ABCF1 LARP4 CASK EIF5B DNA MYO1B CCDC47 EDC3 HNRNPA1 RPN2 PSPC1 TP53 CLIC1 DRG1 PEPD CCAR2 MYH9 JUN AHCTF1 SMS DCUN1D5 CDK9 ZMYM4 MRPL22 PDIA4 NTHL1 MUTYH AP3B1 VRK1 IGF2BP2 HNRNPA0 RAC1 IK PPHLN1 FKBP8 DYNC1H1 NUFIP2 SF1 PFKL RPRD2 MARCKSL1 TNRC6A THOC2 LUC7L NUP93 HK1 NUP153 CFL1 CMTR1 HNRNPL glycosylases PRKAA1 PDCD11 EIF3D RHOC CDC73 PRKACB ESYT2 NUMA1 SNRPD3SNRNP70 NUP98 IGF2BP3 QARS1 CRNKL1 PRPF3 KIF2A TFRC CHERP PYGB PFKM RANGAP1 DNA CDC37 ELAC2EIF2S2 FUBP1 DYNLL1 SPTBN1 PCF11 PKN2 NMT1 PLS1 DIDO1 HSD17B12 CKAP2 PAK4 TXNRD1 EIF2B1 DIS3 KDM4B MRE11 DGCR8 NSF HNRNPM TPT1 KIF4A EDC4 BYSL IRS4 RBM25 MYO1D SNRPD1 COPS2 IQGAP1 CELF1 HNRNPF SF3B1 GOT2 SART1 SNRPD2 NAA15 MORC2 RBBP6 NARS1EIF2S3B HSPA5 nuclease LIMD1 IGF2BP1 SLTM UBAP2L RAVER1 SEC23B MYO6 STAU1 CAPRIN1 TBL2 KRT79NAA25 YTHDF2 SNRPFZC3H14 RRBP1 SNW1 AP2S1 ZNF598 AP3D1 CNOT1 PHF5A PRPF6 WDR33 CSDE1 PRRC2B ANKHD1/ANKHD1-EIF4EBP3 CSNK2A1 UBA2 RAB5C LARP1 LARS1 CPSF7 NAT10 MAML1 SCAF4 EXOSC10 PDE12 CMAS RBM27 SNRPA RBM5 KIF5B ACIN1 CDR2 DNA DIAPH1 RACGAP1PRPF40A GPATCH4 USP39 RBM10 FIP1L1 ZNF638 CSNK1A1 PABPC3COPS3 SRRT PAF1 RBM39 AK2 ESRP2 PRPF4 EXO5 RBM4 HMGA2 PTBP1 CAPN2 GK ABCD3 H1-10 GEMIN5 Cell cycle ADSS2 IFT27 KIF23 ATAD3B PHF3 exonuclease ANLN REEP5 AP2M1 PRKAA2 RGPD4 (includes others) SH3PXD2B ALDH3A2 RC3H2 TRPS1 VPS26A GTF2I ZC3H18 NLE1 HARS2 CEP192 SCAF11 KIF4B The goal is to elucidate the relationship between DDR, iron metabolism, and PCBP1 Several proteins involved in DDR appear as PCBP1 interactors Analysis of the iron chaperone PCBP1 interactome: intersection of DNA repair and iron trafficking

7. The most prominent type of enzymes populating the PCBP1 interactome is the helicases

Functional Enrichment DNA helicases RNA helicases

DDX23 DDX20 DDX56 DDX52 CHD4 HELLS DHX37 DDX10

CHD3 MCM6 1 MOV10 DHX8 DHX36

DDX24 UPF1 MCM5 EP400

DHX16 DDX54

RECQL SMARCA5 Change Fold DHX38 SRCAP 11 CHD1 MCM3 YTHDC2 RBM15 PUF60

ZC3HAV1 DDX18

DHX30 HELZ SNRNP200

Although no Fe-containing helicases were found as part of the PCBP1 interactome, two alternatives are considered: ⦿ Fe-binding motifs are not easily recognized on proteins. Then, some proteins from the PCBP1 interactome may in fact use iron as cofactor. ⦿ These proteins are part of large complexes where another protein is the one requiring iron for function.

What is the role of PCBP1 on DNA repair?