Identification of proteins interacting with the RNAPII FCP1 phosphatase: FCP1 forms a complex with arginine methyltransferase PRMT5 and it is a substrate for PRMT5-mediated methylation.
FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.
Amente
Stefano
S
Department of Genetics, General and Molecular Biology, University of Naples Federico II Naples, via Mezzocannone 8, 80134 Naples, Italy.
Napolitano
Giuliana
G
Licciardo
Paolo
P
Monti
Maria
M
Pucci
Piero
P
Lania
Luigi
L
Majello
Barbara
B
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D006657:Histones / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008745:Methylation; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011496:Protein Methyltransferases / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases; D013379:Substrate Specificity
D006657:Histones; D011496:Protein Methyltransferases; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase