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RNA-seq analysis of 22Rv1 cells with PRMT5, MEP50, pICln knockdown

ABSTRACT: PRMT5 is a methyltransferase that catalyzes symmetric dimethylation of arginine residues in histones (H4R3, H3R8, H3R2, and H2AR3) to regulate transcription of target genes. While PRMT5 is generally considered an epigenetic repressor, recent evidence from our lab and others demonstrate that PRMT5 also functions as an epigenetic activator. Although in vitro biochemical studies suggest that the PRMT5 interacting proteins methylosome protein 50 (MEP50) and methylosome subunit pICln enhance PRMT5 enzymatic activity, how these interacting proteins cooperate with PRMT5 to regulate gene transcription in vivo remains to be elucidated. Here we perform RNA-seq analysis of prostate cancer cells 22Rv1 with knockdown of PRMT5, MEP50, and pICln, in order to elucidate how these proteins control transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154951 | GEO | 2022/05/05

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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RNA-seq analysis of 22Rv1 cells with PRMT5, MEP50, pICln knockdown

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