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. Author manuscript; available in PMC: 2024 Dec 5.
Published in final edited form as: Cell Metab. 2023 Nov 24;35(12):2183–2199.e7. doi: 10.1016/j.cmet.2023.11.001

Figure 3. PRMT1 methylates NPRL2 at R78 residue.

Figure 3.

A, PRMT1, but not SAMTOR, promotes the methylation of the GATOR1 complex. Purified GATOR1 (250 nM) was subjected to in vitro methylation assays for 1 hour in the presence of SAM (1 μM), PRMT1 (100 ng) and SAMTOR (100 ng), and the generation of SAH was analyzed via the MTase-Glo™ Methyltransferase Assay kit.

B, Immunoblotting with the ADMA levels of GATOR1 complex components from A using a panADMA antibody.

C, GST-NPRL2 (wild-type and mutants) were subjected to in vitro methylation analysis as in A.

D, The methylation of NPRL2 (wild-type and mutant) was analyzed with ADMA antibody in cells.

E, Methionine removal inhibited NPRL2 R78me2a methylation. Cells were subjected to anti-Flag immunoprecipitation and analyzed via immunoblotting with NPRL2 R78me2 antibody.

F, The correlation of cytosolic SAM levels, NPRL2 R78me2, and mTORC1 activity (pS6K) from E are presented in F. Data are representative one repeat.

G, NPRL2Flag knock-in MAT2A Dox-off HEK293T cell lines were generated as described in the previous study25. Cells were pretreated with doxycycline (DOX) for 2 days, starved of methionine for 2 hours, and restimulated with methionine (100 μM) or SAM (100 μM) for the indicated time. The WCL and anti-Flag IPs were analyzed via immunoblotting with the indicated antibodies, including a homemade-specific antibody against NPRL2 R78me2a.

H, NPRL2Flag knock-in HEK293T cells expressing tet-on-shPRMT1 were pretreated with DOX for 2 days to knockdown PRMT1, starved of methionine for 2 hours, and restimulated with methionine (100 μM) for 20 min or SAH (100 μM), SAM (100 μM), and Hcy (100 μM) for 6 hours. The WCL and anti-Flag IPs were analyzed via immunoblotting with the indicated antibodies.

I-J, The Km of SAM for human PRMT1 to mediate the methylation of NPRL2 (I) and H4 (J). Purified NPRL2 proteins (10 ng) were incubated with PRMT1 (20 ng) together with the indicated concentrations of SAM, and the level of SAH generation was analyzed as described in A. n=3 biological repeats.

See also Figure S3.