A Novel Methyltransferase (Hmt1p) Modifies Poly(A)⁺-RNA-Binding Proteins

Abstract

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A) ؉ -RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.The primary transcripts of eukaryotic RNA polymerase II, termed heterogeneous nuclear RNAs or pre-mRNAs, undergo a series of processing events in nuclei as they mature into functional, cytoplasmic mRNAs. Prominent among these events are 5Ј-end capping (43), splicing (29, 58), modification of individual bases (15), 3Ј-end cleavage coupled to polyadenylation (30, 37), and export of the processed mRNA to the cytoplasm (20, 35). When pre-mRNAs emerge from the transcription complex and throughout the time they are in nuclei, they are associated with an array of nuclear factors, including heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear RNP particles. Numerous studies have established the roles of small nuclear RNPs in a variety of nuclear processes, such as pre-mRNA splicing (2, 31, 58). However, the functions of hnRNPs have remained unclear; they are proposed to function in nearly all the known steps of mRNA maturation, including pre-mRNA packaging (17, 66), constitutive and alternate splicing (13, 49), and mRNA export (26,42,53).Proteins of partially purified mammalian hnRNP particles contain a high level of the unusual modified amino acid N Protein arginine methyltransferases specific for hnRNP A1 have been identified and partially purified from three different sources: calf brain (28), rat liver (55) and HeLa cells (46). All three enzymes use S-adenosyl-L-methionine (SAM) as the methyl donor and have similar enzymatic properties. Further characterization of these enzymes indicates that their substrates are not limited to pre-mRNA-binding proteins (hnRNPs) (46,51,55). Rather, they have activities toward a wide range of RNA-binding proteins, all of which appear to contain the RGG box RNA-binding motif (6,38).Given the prevalence of this unusual modification in hnRNPs, it is...

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…The GAR domain is found in the nucleolar proteins Nop1/fibrillarin (Schimmang et al+, 1989;Lapeyre et al+, 1990), Gar1p (Girard et al+, 1992), Nsr1p (Lee et al+, 1991), Ssb1p (Clark et al+, 1990), nucleolin (Lapeyre et al+, 1987), gar2 (Gulli et al+, 1995), and in hnRNP proteins such as hnRNP A1, Nop3p/Npl3, and Hrp1p (Beyer et al+, 1977;Kiledjian & Dreyfuss, 1992;Russell & Tollervey, 1992, Birney et al+, 1993Henry et al+, 1996)+ The GAR domain (also called RGG domain) has RNA-helix-destabilizing properties in vitro (Ghisolfi et al+, 1992), is implicated in nonspecific protein-RNA interactions (Burd & Dreyfuss, 1994a, 1994b and in proteinprotein interactions (Cartegni et al+, 1996, Bouvet et al+, 1998)+ So far, however, no phenotype has ever been associated with the removal of the GAR domain(s) of a given protein (Girard et al+, 1994;Sicard et al+, 1998)+ The two GAR domains of the essential Gar1p protein are not required for viability and, consistent with this, Gar1p lacking both GAR domains (Gar1p⌬GAR) accumulates in the nucleolus (Girard et al+, 1994)+ To assess whether removal of the GAR domains of Gar1p results in subtle growth defects, we compared the growth at various temperatures of a gar1-null strain complemented by the gar1⌬GAR allele (JG535-2D-d; see Table 1) with that of the same gar1-null strain complemented by the GAR1 wild-type allele (JG535-2D-c)+ A growth defect is clearly observed for the strain complemented by the mutant allele (Fig+ 2A)+ To determine whether this growth delay is correlated with a pre-rRNA processing defect, we performed pulse-chase labeling experiments with (methyl-3 H)methionine to label methylated RNAs (Fig+ 2B)+ We can detect a slight accumulation of the 32S pre-rRNA and a delay in mature 18S and 25S rRNA production (see Fig+ 1 for a cartoon of the pre-rRNA processing pathway in yeast)+ These data demonstrate that Gar1p lacking its two GAR domains is not fully functional+ Identification of Rrp8p, a novel functional partner of Gar1p, by a synthetic lethal screen, and cloning of RRP8 Deletion of the GAR domains, which partially impairs Gar1p function but still allows cells to grow, could cause synthetic lethality if another component that physically interacts or functionally overlaps with Gar1p becomes mutated+ Thus such components could be identified by searching for the genes that, when mutated in a gar1⌬GAR background, lead to a lethal phenotype+ To screen for such mutations, we used an ade2-ade3-based colony sectoring assay …”

Differential Export Requirements for Shuttling Serine/Arginine-type mRNA-binding Proteins

“…It is comprised of three RNA recognition motifs and one serine/arginine-rich domain with 10 SR or RS motifs. This SR domain contains also two arginine/glycine/ glycine (RGG) motifs, which for Npl3 have been shown previously to be substrates for arginine methylation by the arginine methyltransferase Hmt1 (31)(32)(33)(34).…”

PRMT3 Is a Distinct Member of the Protein Arginine N-Methyltransferase Family

“…The site of arginine methylation for type I enzymes typically occurs within glycine-and arginine-rich (GAR) domains from which similar consensus sequences have been derived (1, 14 -17). Type I enzymes catalyzing these modifications include the yeast Rmt1/Hmt1 catalytic chain (18,19) and the mammalian enzymes containing the catalytic chains PRMT1 (14,20), PRMT3 (15), and CARM1/PRMT4 (21). Compared with the abundant examples of ADMA-containing substrates, the only two known substrates to contain SDMA are myelin basic protein (22,23), and Sm ribonucleoproteins D1 and D3 (24).…”