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 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.

FIGURE 1.

Graphic map of CF I_m59 and CF I_m68 proteins. (A) Domains are indicated by different patterns (see code under C). RRM, RNA recognition motif; Pro-rich, proline–rich domain; GAR, glycine-arginine motif (the box of potential or cryptic GAR is white in CF I_m59 because no methylation was identified); and RS/RD signifies an RS-like domain. Also indicated as insets are included and skipped exons in alternatively spliced forms of CF I_m. (B) The protein sequence of the CF I_m68 fragment in the GST-68_GR clone and with the exon included (in parentheses) in GST-68L1_GR. (C) Fragments of CF I_m59 and CF I_m68 used in the experiments. Protein segments are not drawn to exact scale.

FIGURE 2.

Methylation activity of nuclear and cytoplasmic extracts on recombinant and purified cleavage and polyadenylation factors. Arrowheads indicate locations of corresponding proteins on Coomassie stained SDS gels (0.5–1 μg were loaded). NE, HeLa nuclear extract; CE; HeLa cytoplasmic extract.

FIGURE 3.

CF I_m59 and CF I_m68 subunits are differentially dimethylated. Nuclear extract (NE) or cytoplasmic (CE) extract, purified (CF I_m purif) recombinant P. pastoris expressed CF I_m (rec CF I_m) or partially purified CF II_m (CF II_m part) fractions separated on SDS gels and blotted to NC filters were probed with an α-CF I_m antibody (reacts with both CF I_m59 and CF I_m68 subunits) or α-monomethyl, sym10, or asym24 antibody. The label CF I_m68/L1 points to CF I_m68 (lower band) and CF I_m68_L1 (upper band).

FIGURE 4.

Activity of PRMT1-deficient cell extracts on protein substrates. Cell extracts from PRMT1^(+/+) and PRMT1^(−/−) ES cells were incubated with [¹⁴C]SAM and recombinant GST-68_GRP, PABPN1, or hnRNP K (10 pmol each). Reactions were resolved on 12% SDS PAGE and methylation was detected by autoradiography.

FIGURE 5.

Fractionation of the HeLa cytoplasmic extract on the HiTrap DEAE (A), HiTrap heparin (B), and Superdex-200 (C) gel filtration columns. Fractions were probed on Western blots with antibodies against PRMT5, WD45, pICln, and PRMT1 and an activity assay was performed with substrates GST-68_GR and [³H]SAM. L, load; P, pellet; FT, flow through; and PWI indicates 900 ng of the PRMT5/WD45/pICln complex. The numbers are the column fraction numbers.

FIGURE 6.

The PRMT5/WD45 complex is needed for methylation of the CF I_m68 GAR motif. The control substrate Sm proteins D1/D2 (1 μg) and GST-68_GR (800 ng) were incubated in standard methylation reactions with 1 μL nuclear extract (NE) or cytoplasmic extract (CE) or with increasing amounts of the PRMT5 complexes as indicated. Amounts of enzyme present in the reactions were (from left to right) PRMT5/pICln: 10, 100, and 1000 ng; PRMT5/WD45 and PRMT5/WD45/pICln: 1, 10, and 100 ng.

FIGURE 7.

Only arginines within the GGRGRGRF motif are methylated in CF I_m68. Site directed point mutations within the GAR motif in the GST-68_GR construct and fragments of CF I_m68 were subjected to methylation in vitro by the recombinant PRMT5/WD45 complex. GST-68_GR_3-mut is GST-68_GR with all three arginines (202, 204, and 206) in the GAR motif mutated to alanines. (A) One microgram of the recombinant substrate protein was incubated with 100 ng of PRMT5/WD45 and [³H]SAM. (B) The same as A except that 1 μg of PRMT5/WD45 was used. (C) The same as B except that the reaction contained cold SAM and the blot was probed with the sym10 polyclonal antibody against the SDM arginines. (D) Blot from C stained with Ponceau-S.

FIGURE 8.

In vitro methylation of CF I_m59 and CF I_m68 subfragments. (A) The gels depicted on the middle and right-hand autoradiograms are exposed about 10-fold longer than the autoradiogram to the left. GST-68_GR_3-mut is GST-68_GR with all three arginines (202, 204, and 206) in the GAR motif mutated to alanines. (B) The [³H]SAM activity test with fraction 7 of Superdex-200 column. (C) The [³H]SAM activity test on the native and recombinant substrates. For an explanation of substrate names above the gel see Figure 1 and Supplemental Table 1. Assays were done in the presence of 1.3 pmol of H6-CF I_m68, 1.2 pmol of native CF I_m68, 4.4 pmol of H6-PABPN1, and 4 pmol of native PABPN1. Also included are 4.8 pmol of PRMT1 and PRMT5/WD45. The signal strength normalized to equal amounts of the substrate are indicated as a percent below the gel and the corresponding numbers are indicated by different fonts (normal, bold, and italic).

FIGURE 9.

In vitro activities of recombinant PRMT1 and the PRMT5/WD45 complex. Assays were done in the presence of 2 pmol each of substrates PABPN1, GST-68_GR, or D1/D2 and the reactions were incubated for 2.5 h with 0.32 μM PRMT1 (P1) or the 0.32 μM PRMT5/WD45 complex (P5W) as described in Materials and Methods. (A) For the [³H]SAM assay, 1.04 μCi (or 1.02 μM) [³H]SAM was complemented with an unlabeled SAM to a total concentration of 20 μM. Protein gels were blotted to nitrocellulose and exposed to PhosphorImager screens. (B,C) For Western blots, an unlabeled SAM (20 μM) was present in each reaction. The blots were probed with the sym10 and asym24 antibodies (1:2000 dilution).

FIGURE 10.

Mass spectral analysis of the methylated GAR motif. Sum of the spectra from the LC/MS run of di-, tri-, and quadruply methylated AAFPQGGRGRGRFPGAVPGGDR-peptide with a triple charge.