Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Abstract

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 39 ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 39 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.

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

“…85,2011 ROSCOVITINE INHIBITS EBNA1 2865 late transcription (74,78). In addition, EBNA1 RG1 and RG2 recruitment of PRMT1 and -5 to oriP sites may enhance EBNA1-upregulated RNA export through enhanced RNA 3Ј-terminal cleavage, SM protein effects on splicing, RNA nuclear export, EIF4E and NXF1 binding, and ICP27-like RNA export effects (29,37,51,64,86). Compounds that inhibit or enhance PRMT1 or -5 effects would likely downregulate EBNA1-and oriP-dependent transcription but would also broadly affect protein methylation, RNA processing, and translation.…”

Cleavage factor Imis a key regulator of 3′ UTR length

“…12 Moreover, CF I m 68 from HeLa cells, but not CF I m 59, was found to contain symmetrically dimethylated arginines and that it could be methylated at a glycine-arginine rich (GAR) motif in vitro by the methyltransferase PRMT5. 15 CF I m 59 from HeLa cell nuclei is more strongly modified by asymetrical dimethylation than CF I m 68 and both proteins can be dimethylated by the methyltransferase PRMT1 in vitro mainly at the arginine-rich C-terminus. However, no effects of these modifications on protein-protein interactions or RNA binding capacity of the CF I m factors were so far identified.…”

“…This in turn would also suppress the export and translation of these abortive transcripts because they would lack poly(A) tails. Northern blots with total RNA upon RNAi-mediated knock-down of CF I m 68 appear to show shortening of the transcripts to proximal cleavage sites, 15 although it could still be that long, non-polyadenylated transcripts are unstable.…”

“…In addition, CF I m 59 interacts with the arginine methyltransferase PRMT2. 15,16 By cross-linking and immunoprecipitation (CLIP) followed by deep sequencing we recently mapped the transcriptome-wide binding sites of RNA-binding proteins of the core polyadenylation machinery including CF I m 25, CF I m 59, and CF I m 68. 17 By further quantifying cleavage and polyadenylation (CP) site usage, we could demonstrate that binding of CF I m is predictive for the choice of a polyadenylation site, and that knock-down of CF I m 68 causes a transcriptome-wide increase in proximal CP site use.…”

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia

“…In a general sense, these increases in abundance may be due to increased usage of the respective poly(A) sites, such that more of the transcriptional output is directed toward proximal polyadenylation. Several of the activities of individual polyadenylation complex subunits are affected by phosphorylation, methylation, calmodulin, disulfide remodeling, and ubiquitin (Jacob and Rose, 1984;Thuresson et al, 1994;Colgan et al, 1996;Mizrahi and Moore, 2000;Mouland et al, 2002;Delaney et al, 2006;Ryan, 2007;Addepalli and Hunt, 2008;Ryan and Bauer, 2008;Addepalli et al, 2010;Martin et al, 2010). In addition, alterations in the activities of individual poly(A) complex subunits are often accompanied by large-scale shifts in poly(A) site choice (Jenal et al, 2012;Martin et al, 2012;Thomas et al, 2012;Duc et al, 2013;Li et al, 2015).…”