The function and three-dimensional structure of the oligomeric yeast arginine methyltransferase, Hmt1.

摘要

Arginine methyltransferases catalyze a post-translational modification that has been associated with a wide range of cellular functions including nuclear transport, signal transduction and transcriptional activation. Here we present data for the yeast arginine methyltransferase, Hmt1 (h&barbelow;nRNP m&barbelow;ethylt&barbelow;ransferase 1). Hmt1 is the predominant arginine methyltransferase in yeast and methylates heterogeneous ribnucleoprotein (hnRNPs) that contain RGG, GRG, and RXR consensus sequences. Two of these substrates, Npl3 and Hrp1, are shuttling hnRNPs involved in mRNA processing and export. In the absence of HMT1, both Npl3 and Hrp1 are trapped in the nucleus. In addition, overexpression of Hmt1 enhances the export of Npl3 from the nucleus. A genetic relationship is also demonstrated between HMT1 and a member of the cap-binding complex, CBP80. These data establish a biological role for Hmt1 in the nuclear export of hnRNPs.; One popular model consistent with these results is that methylation may change its substrate's affinity for RNA and, therefore, affect the nature of the ribonucleoprotein complex that exits the nucleus. To examine the effect of methylation on specific RNA-binding, Hmt1 was used to methylate Hrp1 in vitro. Methylated Hrp1 binds to UAUAUA-containing RNAs with the same affinity as unmethylated Hrp1, indicating that methylation does not affect specific RNA-binding. However, RNA itself inhibits the methylation of Hrp1. These data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.; In order to examine whether Hmt1's methyltransferase activity is required for its function, mutations were made in the enzyme's cofactor-binding region. These mutations were unable to catalyze methylation of Npl3 or to restore growth to strains that require HMT1. A cold-sensitive mutation of Hmtl existing outside of the SAM-binding domain, E18V, showed reduced methylation of Npl3, but not other substrates at the non-permissive temperature.; Our crystal structure of Hmt1 determined at 2.9 A resolution reveals that 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. This structure along with mutational data suggests a model for how Hmt1 binds the Npl3 substrate.