Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I.
Mammalian cleavage factor I (CF I(m)) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I(m)25, CF I(m)59, CF I(m)68). It is part of the cleavage and polyadenylation complex responsible for processing the 3' ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3' processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I(m)68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I(m)68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I(m)68 we could exclusively identify arginines in a GGRGRGRF or "GAR" motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I(m)68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I(m)59 and CF I(m)68. The results suggest that native-as compared with recombinant-protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I(m) methylation in the context of RNA export is discussed.
Martin
Georges
G
Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Ostareck-Lederer
Antje
A
Chari
Ashwin
A
Neuenkirchen
Nils
N
Dettwiler
Sabine
S
Blank
Diana
D
Rüegsegger
Ursula
U
Fischer
Utz
U
Keller
Walter
W
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008322:Mammals / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*
D012322:RNA Precursors; D005340:Fibrinogen; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases