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Rose, AM, Belford, HG, Shen, WC, Greer, CL, Hopper, AK and Martin, NC (1995) Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase. Biochimie 77:45-53
Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway. Indirect immunofluorescence experiments using antibody to N2,N2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells. Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p. Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease. Cells overproducing Trm1p also concentrate it to the nuclear periphery. Thus, the site(s) of interaction of Trm1p are not easily saturable and are likely to be in excess to Trm1p. Trm1p is shared by mitochondria and the nucleus. Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme. Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.
Amino Acid Sequence; Blotting, Western; Cell Fractionation; Cell Nucleus/enzymology; Cytosol/enzymology; Deoxyribonucleases/metabolism; Endoribonucleases/analysis; Fluorescent Antibody Technique; Membrane Proteins/analysis; Membrane Proteins/genetics; Mitochondria/enzymology; Molecular Sequence Data; Mutation/genetics; Nuclear Envelope/enzymology; Nuclear Pore Complex Proteins; Nuclear Proteins/genetics; Phosphoric Diester Hydrolases/analysis; Polynucleotide 5'-Hydroxyl-Kinase/analysis; Polynucleotide Ligases/analysis; RNA, Fungal/biosynthesis; RNA, Transfer/biosynthesis; Saccharomyces cerevisiae/enzymology; Sodium Chloride/pharmacology; tRNA Methyltransferases/analysis; tRNA Methyltransferases/metabolism
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