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Small-molecule inhibition of PRMT5 activates p53 in erythropoiesis through perturbation of mRNA processing, transport and protein translation

ABSTRACT: The protein arginine methyltransferase PRMT5 catalyzes symmetric di-methylation of numerous proteins involved in epigenetic gene regulation, mRNA splicing and DNA repair. Several small molecule inhibitors of PRMT5 are in clinical trials for acute myeloid leukemia, myelodysplasia, lymphoma and metastatic solid tumors. Here, we report the development of a new potent and specific small molecule inhibitor of PRMT5 (CTX-034) that binds and competes for the arginine substrate binding pocket. CTX-034 suppresses erythroid progenitor growth 30-fold more potently than myeloid progenitor growth through activation of p53. Using a combination of RNA-seq and proteomics, we show that PRMT5 binds and methylates numerous proteins required for protein translation including RNA transport (Heterogeneous Nuclear Ribonucleoprotein A1), initiation of translation (Poly(A) Binding Protein Cytoplasmic 1) and ribosomal assembly (Ribosomal Protein S10). Using an in vivo mouse model of JAK2V617F-driven Polycythemia Vera, CTX-034 suppresses the formation of immature erythroblasts as effectively as the JAK inhibitor Ruxolitinib.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE145285 | GEO | 2024/02/01

REPOSITORIES: GEO

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Project description:The protein arginine methyl transferase PRMT5 is an enzyme expressed in oligodendrocyte lineage cells and responsible for the symmetric methylation of arginine residues on histone tails. Previous work from our laboratory identified PRMT5 as critical for myelination, due to its transcriptional regulation of survival and early stages of differentiation. However, besides its nuclear localization, PRMT5 is found at high levels in the cytoplasm of oligodendrocyte progenitor cells (OPCs) and yet, its interacting partners and non-histone substrates in this lineage, remain elusive. By using mass spectrometry on protein eluates from extracts generated from OPC and immunoprecipitated with PRMT5 antibodies, we identified 1116 proteins as PRMT5 interacting partners. These proteins are related to molecular functions such as RNA binding, ribosomal structure, nucleotide binding, cadherin and actin binding, GTP and GTPase activity. We then investigated PRMT5 substrates using iTRAQ-based proteomics on protein samples isolated from CRISPR-PRMT5 knockdown immortalized oligodendrocyte progenitors compared to CRISPR-EGFP controls. This analysis identified 169 peptides with statistically different symmetric methylation of arginine residues in the two groups. Those peptides led to 30 unique non-histone substrates of PRMT5. The majority of these proteins were consistent with PRMT5 substrates, previously identified in distinct cancer cell lines (e.g. the transcription factor ZN326), and were functionally related to RNA processing and transport, splicing, regulation of mRNA stability and transcription. In addition, we detected three substrates as unique to the oligodendrocyte lineage and not identified in cancer cells. They included the proline-rich protein PRC2C, involved in methyl-RNA binding, the RNA binding protein KHDR2, regulating splicing events and the heterogeneous nuclear RNA binding protein HNRPD, involved in regulation of RNA stability. Together, these results highlight a cell-specific role of PRMT5 in regulating, not only transcription, but RNA processing and translation in OPCs. (287 WORDS)

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Small-molecule inhibition of PRMT5 activates p53 in erythropoiesis through perturbation of mRNA processing, transport and protein translation

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