NF90/NFAR (nuclear factors associated with dsRNA) – a new methylation substrate of the PRMT5-WD45-RioK1 complex

NF90 interacts with the PRMT5/WD45/RioK1 complex

In previous work, NF90 was identified as a new potential interaction partner of the PRMT5/WD45/RioK1 complex by mass spectrometry (Guderian et al. 2011). In this follow-up study, we show that NF90 is indeed a binding partner and a new substrate of PRMT5 in vitro and in vivo. To investigate the complex composition and the recruitment of NF90, we generated cell lines, stably overexpressing PRMT5, WD45, pICln, and RioK1 as GFP fusion proteins. By immunoprecipitation studies, we observed co-immunoprecipitation of NF90 with GFP-PRMT5, GFP-WD45, and GFP-RioK1, whereas we could not precipitate NF90 with GFP-pICln (Figure 1A). Data by Guderian et al. showed, that the substrate specificity and recruitment of new substrates of PRMT5 are controlled by its adaptor proteins RioK1 or pICln (Guderian et al. 2011). NF90, predominantly purified by GFP-RioK1 but not by pICln, supports this model of regulating the mutual substrate specificity of PRMT5. Further pulldown experiments, utilizing heterologous expressed GST fusion proteins from Escherichia coli, confirmed these results: endogenous NF90 from HEK293 lysates bound to GST-RioK1 and GST-PRMT5 but not to GST-pICln (Figure 1B).

Figure 1:

NF90 interacts with the PRMT5/WD45/RioK1 complex. (A) Immunoprecipitation from GFP-PRMT5, -WD45, -Riok1, -pICln, and GFP overexpressing cells. Protein expression was induced with 0.1 μg/mL doxycycline for 24 h and as input 25 µg of total protein was loaded. After cell lysis, GFP-IP was performed and analyzed by Tris/glycine-SDS-PAGE and Western blotting, using antibodies against GFP and NF90. NF90 was co-immunoprecipitated with GFP-PRMT5, GFP-WD45 and GFP-RioK1. (B) Pulldown assays with recombinant GST-PRMT5, -WD45, -pICln, -RioK1, and GST purified from E. coli were executed in HEK293 lysate overnight at 4 °C. Co-precipitation of NF90 was analyzed using NF90 antibody and was detectable for GST-RioK1 and GST-PRMT5. (C) Pulldown assay as described above with different truncated forms of GST-NF90 purified from E. coli. Detection of co-precipitated proteins was performed with RioK1, WD45, and PRMT5 antibodies. Only the C-terminus of NF90 interacts with RioK1, WD45, and PRMT5. (D) Interaction studies using microscale thermophoresis measurements (MST). GST-PRMT5 was labeled with AlexaFluor488 fluorescent dye and measured against the interaction partners RioK1, WD45, and NF90. GST-PRMT5 showed a high affinity to its substrate NF90 with 57 nM. WB: Western blotting.

Interaction with the PRMT5/WD45/RioK1 complex occurs via the C-terminus of NF90

NF90 is a multi-domain protein consisting of two double-stranded RNA-binding motifs (DRBM), a domain associated with zinc fingers (DZF), a bipartite nuclear localization signal (NLS), and an RG-motif (see also Figure 2 C). Due to the multi-domain structure of NF90, we generated truncated forms of NF90 (Figure 2C) to characterize the interaction of NF90 with the proteins of the PRMT5/WD45/RioK1 complex in more detail (Figure 1C). To this end truncations of NF90 were generated by dividing the two DRBMs (NF90 aa1-479 and NF90 aa480-702) and also dividing between the DZF domain and both DRBMs (NF90 aa1-391 and NF90 aa392-702) (Figure 2C). Another truncated form represents NF90 without the RG-motif in the C-terminal region (NF90 aa1-639) (Figure 2C). Interaction studies based on these forms of NF90 revealed an interaction of the PRMT5/WD45/RioK1 complex with the C-terminal region of NF90 spanning from amino acid 392 to 702 (Figure 1C). Interestingly, the truncated form aa1-639, which represents almost the entire protein except for the RG-motif, showed only weak interaction. This indicates that the interaction predominantly occurs in the RG-rich region at the C-terminus of NF90 (Figure 1C and 2C). To determine the affinities among the components of the PRMT5 complex and with the substrate protein NF90, microscale thermophoresis experiments were performed. For this purpose, GST-PRMT5 was labeled with Alexa flour 488 fluorescent dye and first measured against the known interaction partners of the complex, RioK1 and WD45. PRMT5 showed a strong affinity for RioK1 with a K_D of 100 nM and still a strong attachment to WD45 with a K_D of 622 nM (Figure 1D, Supplementary Figure 1). In contrast, the affinity of the substrate protein NF90 towards the methyltransferase PRMT5 was determined with a high affinity of 57 nM (Figure 1D, Supplementary Figure 1). Only marginal changes of affinity could be observed if WD45 was added or WD45 and RioK1 in combination. The data so far lead to the assumption that NF90 is a strong binding partner of PRMT5 in vitro and that substrate binding directly occurs to PRMT5 by the RG-rich stretch of NF90.

Figure 2:

The C-terminus of NF90 is methylated by PRMT5. (A) Radioactive in vitro methylation assay using GST-NF90 purified from E. coli and 200 ng recombinant PRMT5 and 1 µCi [3H]-SAM was performed with and without 1 mM PRMT5 inhibitor EPZ015666. Samples were separated by Tris/glycine-SDS-PAGE and radioactive incorporation was analyzed by Western blotting and autoradiography. The addition of 1 mM PRMT5 inhibitor EPZ015666 inhibits the methylation of NF90 by PRMT5. (B) Investigation of the time-dependent methylation of NF90. 880 ng NF90 purified from E. coli without GST-tag was incubated with 200 ng recombinant PRMT5 and 1 µCi [3H]-SAM. The reaction was terminated by addition of SDS sample buffer and the samples were analyzed as described in (A). (C) Schematic overview of the domains and truncated forms of NF90 used in this work. The asterisks indicate methylatable forms and the distinctive RG-boxes have been highlighted in yellow. (D) Methylation assay of truncated NF90 forms purified from E. coli as described in (A). NF90 is methylated in the C-terminus between amino acids 640 and 702 by PRMT5. AR: autoradiography, AB: amido black staining.

NF90 is a new substrate of PRMT5

Based on this strong in vitro interaction, we performed radioactive in vitro methylation assays with recombinant NF90 and active PRMT5 to test whether NF90 is methylated by PRMT5. Indeed, the radioactive methyl group from [3H]-SAM was transferred to GST-NF90 (Figure 2A). Treatment with the PRMT5-specific inhibitor EPZ015666 did not result in radioactive incorporation (Chan-Penebre et al. 2015). Both experiments prove that NF90 is symmetrically methylated by PRMT5 in vitro (Figure 2A).

To assess the kinetics of NF90 methylation, we performed a titration of the methylation reaction. As evident in Figure 2B, a saturation of NF90 methylation did occur already within 10 min. To gain a more detailed view on the methylation dynamics of NF90, 2 min steps, up to 24 min were performed. Again, no increase in methylation could be observed after 12 min (Figure 2B). The NF90 protein contains several putative methylation sites (arginine-glycine-repeats, RG-Box) in the C-terminus between the amino acids 609 to 656. To further determine the location of the methylation sites within the NF90 protein we analyzed truncated forms (Figure 2D) by methylation assays. We identified eight arginine residues as potential targets in this region (Figure 2C, highlighted in yellow), in particular, we observed methylation of the C-terminal forms NF90 aa392-702 and NF90 aa480-702 and the wild-type NF90 protein (Figure 2D). In contrast, no methylation was observed for the N-terminal forms NF90 aa1-391, NF90 aa1-479, and NF90-aa1-639 (Figure 2D). These results clearly show methylation of NF90 by PRMT5 exclusively is located within the C-terminal RG-rich sequence ranging from amino acid 640 to 702.

NF90 is methylated in vivo by PRMT5 in an RG-rich region in the C-terminus

Based on these data, we focused on the RG-motif in the C-terminal region of NF90 in more detail. This RG-rich region contains seven glycine-arginine residues that potentially may serve as methylatable residues to PRMT5 (Figure 2C). All arginines in the RG-rich region of NF90 at positions 640, 642, 644, 649, 651, 653, and 655 were exchanged to the structurally similar but non-methylatable lysines. The obtained methylation deficient mutant (NF90-7x R-to-K) and the NF90 wildtype protein were expressed in E. coli and the purified recombinant proteins were subsequently analyzed by methylation assays (Figure 3A). In contrast to wild-type NF90, no methylation could be detected in the methylation-deficient mutant, indicating that the methylation sites are located in the region of amino acids 640 to 655 in the C-terminus of NF90.

Figure 3:

NF90 arginine mutants and in vivo inhibitor studies. (A) Recombinant NF90 wt, and NF90 7x from E. coli was incubated with 200 ng recombinant PRMT5 and 1 µCi [3-H]-SAM. Samples were separated by Tris/glycine-SDS-PAGE and radioactive incorporation was analyzed by Western blotting and autoradiography. In the NF90 7x mutant, all seven potential arginine methylation sites are mutated to lysines (see Figure 2C). PRMT5 was not able to methylate the sevenfold arginine to lysine mutant. (B) HEK293 cells were treated with 20 µM of the S-adenosylmethionine-dependent methyltransferase inhibitor adenosine dialdehyde (Adox). 500 µg lysate with 1 µg NF90 antibody or as control Preimmunserum (PIS) and Protein G Sepharose was incubated for 1.5 h and immunopurified NF90 was subjected to a methylation assay as described in (A). Only NF90 from Adox-treated cells could be methylated in vitro. (C) Flp-In T-REx 293 cells stably expressing GFP-NF90 wt and GFP-NF90 7x were stimulated with 0.1 μg/mL doxycycline for 24 h and treated with Adox as described in (B). After GFP-IP, a methylation assay was performed. No methylation of purified GFP-NF90 occurred in control cells (DMSO), whereas NF90 immunopurified from cells treated with Adox could be methylated in vitro. The sevenfold lysine mutant GFP-NF90 7x showed no methylation in all conditions. AR: autoradiography, AB: amido black staining, WB: Western blotting.

Next, we investigated the intracellular methylation status of endogenous NF90 from HEK cells. For this purpose, we immunoprecipitated endogenous NF90 from HEK cells and used it as a substrate for an in vitro methylation assay. However, endogenous NF90 showed no incorporation of radioactive 3H (Figure 3B), leading to the assumption, that NF90 is already completely methylated in vivo. To substantiate this hypothesis, HEK293 cells were treated with 20 µM adenosine dialdehyde (Adox) to disable endogenous methylation, before the respective cell extract was subjected to immunoprecipitation of NF90. Adox is a well-known broad inhibitor of methyltransferases (Chen et al. 2004). In consequence, newly synthesized proteins remain hypomethylated upon Adox treatment (Chen et al. 2004) and therefore are receptive for in vitro methylation if purified from the respective extracts. By using immunoprecipitated endogenous NF90 with and without treatment by Adox, we clearly could show methylation by Adox treated NF90, but not by DMSO/control-treated NF90 upon incubation with recombinant PRMT5 (Figure 3B). To this point, treatment of HEK293 cells with Adox does not alter NF90 cellular expression nor efficiency of immunoprecipitation (Figure 3B) but does alter endogenous methylation status of NF90. Upon the observed rate of methylation of Adox pretreated NF90 substrate protein, endogenous NF90 probably is fully methylated in the cell under normal conditions. To prove this finding, we repeated the experiments with HEK293 Flp-In T-REx cell lines inducibly overexpressing NF90 wildtype as a GFP-fusion protein (GFP-NF90 wt) or the methylation-deficient GFP-NF90 7x mutant (Figure 3C). First, the intracellular protein methylation was blocked by Adox treatment, and subsequently, NF90 protein expression was induced. GFP-immunoprecipitations allowed for exclusive precipitation and measurement of newly synthesized NF90, excluding endogenous, untagged NF90. Again, no methylation of purified GFP-NF90 occurred in control cells (DMSO), whereas NF90 from Adox treated cells could be methylated in vitro (Figure 3C). Taken together, these experiments indicate that endogenous as well as overexpressed NF90 are completely methylated in the normal cellular setting.

Cloning and plasmids

NF90 (CCDS12247.1) cDNA was generated from HEK293 cells with cDNA Transcription Kit (4,368,814, Applied Biosystems) and cloned into pGEX-6P-1 (28,954,648, Cytiva) and pcDNA5-FRT-TO (V601020, Invitrogen) using Gibson assembly (NEB, E2621S) or restriction enzyme cloning. Phusion Polymerase (M0530, NEB) and the following primers (Sigma-Aldrich) were used:

pGEX-6P-1-NF90 wt, 5′-GTGAATTCATGCGTCCAATGCGAATT-3′

and 5′-GTGCGGCCGCCTAGGAAGACCCAAAATCATGAT-3’;

pGEX-6P-1-NF90AA1-639,

5′-CACAAGAGGAGCTGGAGGCAGTCCAGAACATGGTG-3′

and 5′- TCCAGCTCCTCTTGTGTTGGATAAACGGAAGAATG-3′

and 5′-AACCTTTAGGCGGCCGCATCGTGACTGACTG-3′

and 5′-CGGCCGCCTAAAGGTTGGGGGGTGGGGGCAC-3’;

pGEX-6P-1-NF90 AA1-479, 5′-GTGAATTCATGCGTCCAATGCGAATT-3′

and 5′-GTGCGGCCGCCCCCTTGCTCGAGTCC-3’;

pGEX-6P-1-NF90 AA480-702, 5′-GTGAATTCGAGGACTCGGCTGAGGAG-3′

and 5′-GTGCGGCCGCCTAGGAAGACCCAAAATCATGAT-3’;

pGEX-6P-1-NF90 AA1-391, 5′-GTGAATTCATGCGTCCAATGCGAATT-3′

and 5′-GTGCGGCCGCCTACTGAATCTTCTTCTTCTTTTTGCTG-3’;

pGEX-6P-1-NF90 AA392-702, 5′-GTGAATTCAAGAAAGAGGAGAAGGCAGAG-3′

and 5′-GTGCGGCCGCCTAGGAAGACCCAAAATCATGAT-3’;

pGEX-6P-1-NF90 AA609 R->K, 5′-AGCCCCAGTACCCGTCAAAGGGGGACC-3′

and 5′-GGTCCCCCTTTGACGGGTACTGGGGCT-3’;

pGEX-6P-1-NF90 7x AA640, 642, 644, 649, 651, 653, 655 R->K (GeneArt Gene Synthesis, Invitrogen);

pGEX-6P-1-NF90 AA640 R->K,

5′-CCCCAACCTTAAAGGGCGGGGAAGAGGCGGGAG-3′

and 5′-CCCCGCCCTTTAAGGTTGGGGGGTGGGGGCAC-3’;

pGEX-6P-1-NF90 AA642 R->K,

5′-TCGAGGGAAGGGAAGAGGCGGGAGCATCCGGGGAC-3′

and 5′-CCGCCTCTTCCCTTCCCTCGAAGGTTGGGGGGTGG-3’;

pGEX-6P-1-NF90 AA644 R->K,

5′-TCGAGGGCGGGGAAAAGGCGGGAGCATCCGGGGAC-3′

and 5′-CCGCCTTTTCCCCGCCCTCGAAGGTTGGGGGGTG-3’;

pGEX-6P-1-NF90 AA649 R->K,

5′-AGGCGGGAGCATCAAGGGACGAGGGCGCGGGCGAG-3′

and 5′-CGTCCCTTGATGCTCCCGCCTCTTCCCCGCCCTC-3’;

pGEX-6P-1-NF90 AA651 R->K,

5′-ATCCGGGGAAAAGGGCGCGGGCGAGGATTTGG-3′

and 5′-CCCGCGCCCTTTTCCCCGGATGCTCCCGCCTC-3’;

pGEX-6P-1-NF90 AA653 R->K,

5′-ACGAGGGAAGGGGCGAGGATTTGGTGGCGCCAAC-3′

and 5′-AATCCTCGCCCCTTCCCTCGTCCCCGGATGCTCC-3’;

pGEX-6P-1-NF90 AA655 R->K,

5′-GGGCGCGGGAAAGGATTTGGTGGCGCCAACCATGG-3′

and 5′-ACCAAATCCTTTCCCGCGCCCTCGTCCCCGGATG-3’.

Protein expression and purification

E. coli BL21 was transformed and grown overnight at 37 °C on selection plates (100 μg/mL ampicillin). 150 mL LB medium was inoculated and grown overnight at 37 °C. 1 L SB media (35 g/L Tryptone, 20 g/L Yeast extract, 5 g/L NaCl) culture was grown to an OD₆₀₀ of 0.8. Protein expression was induced with 1 mM IPTG at 18 °C overnight. The bacteria were lysed in 300 mM Na

Cl, 50 mM Tris pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.01% (v/v) Igepal, protease inhibitors (cOmplete Protease Inhibitor, 4693,132,001, Roche) and 50 mg/mL lysozyme. After sonication, lysates were centrifuged for 30 min at 10,000 g, incubated for 1.5 h at 4 °C with Glutathione-Sepharose 4B (Cytiva, 17,075,601), and washed three times with lysis buffer. The GST-tag was cleaved with PreScission Protease (Cytiva, 27,084,301) overnight at 4 °C.

The active PRMT5/WD45 complex was purchased from Active Motif (31,521). GFP-tagged proteins were expressed in HEK293 cells and purified by GFP-Trap Agarose (gta-20, Chromotek).

Antibodies

Primary antibodies: α-NF90 (A303-651A, Bethyl), α-PRMT5 (2252, CST), α-WD45 (2823, CST), α-RioK1 (NBP1-30103, Novus Biologicals), α-pICln (sc-393525, Santa Cruz), α-GFP (3H9, Chromotek). Secondary antibodies using LI-COR Odyssey Imaging System: IRDye 680LT goat α-rabbit, IRDye 680LT goat α-mouse, IRDye 800CW donkey α-rabbit, IRDye 800CW donkey α-mouse, IRDye 800CW goat α-rat (LI-COR Biosciences).

Cell culture and cell lines

HEK293 cells were cultured in high glucose DMEM (41,965,039, Gibco) with 10% (v/v) tetracycline-free FCS (10,270,106, Gibco) and 100 units/mL of Penicillin and 100 μg/mL Streptomycin (15,140,122, Gibco) in a 5% CO₂ atmosphere at 37 °C. Cells were washed with PBS (14,190,169, Gibco) and treated with Trypsin-EDTA (25,300,054, Gibco).

Inducible Flp-In T-REx 293 cell lines (R78007, Invitrogen), stably expressing NF90 wt, NF90 7x, GFP-NF90 wt, GFP-NF90 7x, GFP-PRMT5, GFP-WD45, GFP-pICln, and GFP-RioK1 were generated by co-transfecting 4.5 µg pOG44 and 0.5 µg pcDNA5 plasmids with FuGENE HD (E2311, Promega). Cells were selected with 200 μg/mL Hygromycin B Gold (ant-hg-1, Invivogen) and 5 μg/mL Blasticidin (ant-bl-05, Invivogen) for three weeks. GFP-pICln cells were generated as described in (Schmitz et al. 2021). Protein expression was induced with 0.1 μg/mL Doxycycline and cells were harvested after 24 h. For methylation inhibition 20 µM of the S-adenosylhomocysteine hydrolase inhibitor Adenosine Dialdehyde (Adox) (Cay15644, Cayman Chemical) was used.

Immunoprecipitation and immunoblotting

Cell lysates were generated using lysis buffer with 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and 1x Protease inhibitor (P2714, Sigma–Aldrich). Protein concentration was measured by Bradford assay (5,000,006, Bio-Rad). 500 μg cell lysate was used for immunoprecipitation together with 1 µg of antibodies and Protein G Sepharose (17,061,801, Cytiva) at 4 °C for 1.5 h with rotation. As input 25 µg of total protein was loaded. GFP-tagged proteins were purified by GFP-Trap Agarose (gta-20, Chromotek) at 4 °C for 1.5 h with rotation. Samples were washed three times with washing buffer (lysis buffer without Triton X-100 and protease inhibitors) and eluted in sample buffer (375 mM Tris pH 7.5, 25.8% (w/v) glycerol, 12.3% (w/v) SDS, 0.06% (w/v) bromophenol blue, 6% (v/v) β-mercaptoethanol, pH 6.8). Subsequently, samples were separated by Tris/Glycine-SDS-PAGE and transferred to a PVDF membrane (Immobilon-FL, Merck Millipore). The immunoblot detection was carried out using the indicated primary and fluorescent-labeled secondary antibodies and the LI-COR Odyssey Imaging System.

In vitro methylation

Target proteins were incubated with 150 ng of Flag-PRMT5/WD45 from Sf9 cells (Active Motif, 31,521) and 1 µCi Adenosyl-L-Methionine, S-[methyl-3H] (Hartmann-Analytic, ART0288) in 50 mM Tris pH 7.5, 1 mM EGTA and 1 mM EDTA for 1.5 h at 37 °C. The reaction was stopped by adding sample buffer. Samples were separated by Tris/Glycine-SDS-PAGE and, after blotting and amido black staining (40% Methanol (v/v), 10% Acetic acid (v/v), 0.1% Amido black 10B (w/v)), analyzed by autoradiography with Amersham Hyperfilm MP (28,906,844, Cytiva) and BioMax TranScreen LE (1,622,034, Carestream).

Microscale thermophoresis

A Monolith NT 115 (NanoTemper Technologies) was used for microscale thermophoresis binding studies. Purified recombinant proteins GST-PRMT5, RioK1 or NF90 were labeled with AlexaFluor488-NHS (A20000, Invitrogen) in 50 mM HEPES, 300 mM NaCl, pH 7.5. 50 nM of labeled proteins and 4–14 µM of unlabeled proteins were used for interaction studies and measured as triplicates with 50% MST power and 50% LED power in premium or hydrophobic capillaries.