The essential yeast RNA binding protein Np13p is methylated

Abstract

Arginine methylation is a prevalent modification found in many RNA binding proteins, yet little is known about its functional consequences. Using a monoclonal antibody, 1E4, we have shown that the yeast NPL3 gene product Np13p, an essential RNA binding protein with repeated RGG motifs, is arginine-methylated in vivo. The 1E4 epitope can be generated by incubating recombinant Np13p with partially purified bovine arginine methyltransferase block this reaction. Np13p methylation requires S-adenosyl-L-methionine and also occurs in yeast extracts. An Np13p deletion mutant lacking the RGG domain is not a substrate for methylation, suggesting that the methylation sites lie within the RGG motifs. The discovery of arginine methylation in a genetically tractable organism provides a powerful entrée to understanding the function of this modification, particularly in view of the many roles postulated for Np13p in RNA processing and transport. The recent discovery of phosphorylated serine residues within the RGG domain suggests a hypothesis in which a molecular switch governed by methylation and phosphorylation regulates the biochemical properties of the Np13p RGG domain.

Figure 1

Schematic diagram of the Npl3 protein domains. The Npl3 protein, represented as a rectangle with the N terminus at left and the C terminus at right, has an N-terminal domain of unknown function, two central RRMs, and a C-terminal RGG box RNA binding domain that overlaps with an SR domain containing eight SR or RS dipeptides. The sequence of this SR/RGG domain is listed below, with the RS, SR, and RGG peptides in larger font and the SRGG tetrapeptides underlined. One or more serine residues within the SR or RS dipeptides appear to be phosphorylated and one or more arginine residues within the RGG tripeptides appear to be methylated (see text).

Figure 5

Npl3p lacking the C-terminal domain is expressed in vivo without the mAb 1E4 epitope. Duplicate extracts from cells carrying either a plasmid expressing an Npl3p deletion mutant lacking the C-terminal domain (ΔRGG) (lanes 1 and 2) or a control plasmid lacking an insert (lanes 3 and 4) were examined by immunoblot analysis using anti-Npl3p polyclonal antibodies (A) or mAb 1E4 (B). WT and ΔRGG on the left mark the positions of full-length Npl3p expressed from the wild-type chromosomal gene and the deletion mutant expressed from the plasmid, respectively.

Figure 2

mAb 1E4 recognizes a modified epitope on Npl3p. Duplicate immunoblots were probed with anti-Npl3p mAbs 2F1 (A) or 1E4 (B). Lanes 1–3 and 7–9 contain 0.25, 0.5, or 1.0 μl of whole-cell yeast extract (approximately 10 mg of total protein per ml), respectively. Lanes 4–6 and 7–9 contain 12.5, 25, or 50 ng of recombinant Npl3p (rNpl3p) expressed in bacteria and purified exploiting a six-histidine tag that also increases the molecular mass of the protein relative to untagged Npl3p expressed in yeast (yNpl3p).

Figure 3

Arginine methyltransferase generates the mAb 1E4 epitope on rNpl3p. The mAb 1E4 reactivity of 1 μg of purified rNpl3p treated in vitro with a bovine arginine methyltransferase in the presence of the methyl donor SAM was examined by immunoblot analysis. Lanes: 7, 11, and 15, rNpl3p methylation reactions performed in parallel; 1–3, reactions lacking the methyl donor, enzyme, or substrate, respectively; 4–6, 8–10, and 12–14, reactions that included the indicated micromolar concentrations of the competitor peptides listed at the bottom.

Figure 4

Arginine methyltransferase and yeast extract transfer a methyl group from SAM to rNpl3p. Methylation of purified rNpl3p was analyzed by fluorography after denaturing gel electrophoresis of rNpl3p that had been incubated with a bovine arginine methyltransferase (A) or yeast extract (B) in the presence of SAM carrying a tritiated methyl group. Labels above the lanes are as in Fig. 3.

Figure 6

Npl3p lacking the C-terminal domain is not a substrate for methylation in vitro. Purified recombinant full-length Npl3p (WT) or truncated Npl3ΔRGGp (ΔRGG) were tested as substrates for methylation in vitro by the bovine arginine methyltransferase or yeast extract as in Fig. 4. Methylation was assayed by fluorography (B) of the gel stained for protein with Coomassie blue (A) (dots mark the locations of the WT and ΔRGG substrates). Assays were performed without substrate (lanes 1 and 5), with either substrate separately (lanes 2, 3, 6, and 7), and with the substrates together (lanes 4 and 8).