The structure and oligomerization of the yeast arginine methyltransferase, Hmt1.

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.

Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function. Cofactor binding and substrate interactions.

Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues. The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth. However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function. We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo. Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1. In addition these mutations affect nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C. These results define new aspects of Hmt1 and reveal the importance of its activity in vivo.

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation.

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Arginine methylation facilitates the nuclear export of hnRNP proteins.

Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated on arginine residues. In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p. Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export. Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A)+ RNA, however, is not affected by the lack of methylation. Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80). Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus.

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.