A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

Schimmang, Thomas; Tollervey, David; Kern, Hildegard; Frank, Rainer; Hurt, Ed

doi:10.1002/j.1460-2075.1989.tb08584.x

Cited by 303 publications

(302 citation statements)

References 60 publications

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“…In vitro translated, 35 S-labeled hU3-55k was synthesized using wheat germ extract (TNT Coupled Wheat Germ Extract System, Promega) in the presence of translation grade [ 35 S]methionine (1000 Ci/mmol; Amersham, NJ). Approximately 5% of the biotinylated RNA made in a 25 µl transcription reaction was added to 10 µl of in vitro translated hU3-55k, 40 U RNasin (Promega), 2 µg non-specific competitor yeast tRNA (Boehringer Mannheim, IN), and 2 µl of 10× binding assay buffer (100 mM Tris, pH 8.0, 1 M KCl and 0.5% NP-40).…”

Section: Analysis Of U3-55k Association With U3 Snorna In Vitromentioning

confidence: 99%

“…The RNA is associated with proteins common to all Box C/D snoRNAs, including fibrillarin/Nop1p (34,35), Nop56p (36) and Nop58/Nop5p (37)(38)(39) and proteins specific to the U3 snoRNP, including Sof1p (supressor of fibrillarin; 40), Mpp10 (41,42), Lcp5p (43), Imp3p (44), Imp4p (44) and U3-55k (29,45). Each of these proteins (like U3 RNA) are essential and have been shown to be specifically required for the maturation of 18S rRNA [39][40][41]43,44,46,47 personal communication].…”

Section: Introductionmentioning

confidence: 99%

“…U3-55k belongs to the WD repeat family of proteins (45), characterized by the core sequence [GH]-(X [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] )- [WD] arranged in repeats of four to eight units (48)(49)(50). β-Transducin (Gβ), a member of the same family, has seven WD repeats which fold into a propeller-like structure with each repeat motif forming a discrete blade of the propeller (51)(52)(53).…”

Section: Introductionmentioning

confidence: 99%

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Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

Lukowiak

Granneman²,

Mattox

et al. 2000

Nucleic Acids Research

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U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. U3-55k is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the U3-55k protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of U3-55k with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by U3-55k in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis U3-55k protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of U3-55k resulted in loss of association with U3 RNA and also loss of localization of U3-55k to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of U3-55k in vivo.

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Section: Analysis Of U3-55k Association With U3 Snorna In Vitromentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

Lukowiak

Granneman²,

Mattox

et al. 2000

Nucleic Acids Research

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“…As a control for the temperature-sensitive mutants, we tested semi-intact cells prepared from a temperaturesensitive mutant in the NOP1 gene (Schimmang et al, 1989). Nopl is a nucleolar protein important for maturation of ribosomes (Tollervey et al, 1993).…”

Section: A Role For Nucleoporins In Nuclear Transport In Semi-intact mentioning

confidence: 99%

Reconstitution of nuclear protein transport with semi-intact yeast cells.

Schlenstedt

Hurt

Doye

et al. 1993

The Journal of Cell Biology

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Abstract.We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLScontaining proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nspl inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nspl mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.M ANY nuclear-destined proteins contain short stretches of amino acids (termed NLS ~ for nuclear localization sequences) that target the protein to the nucleus (for review see Silver, 1991;Goldfarb and Michaud, 1991;Nigg et al., 1991;Garcia-Bustos et al., 1991a). The process of nuclear protein uptake occurs in at least two steps; NLS-dependent binding at the nuclear envelope followed by ATP-dependent translocation through the nuclear pore complex (Newmeyer and Forbes, 1988;Richardson et al., 1988;Akey and Goldfarb, 1989). Because of the apparent saturability (Goldfarb et al., 1986) and specificity of NLS function (e.g., Kalderon et al., 1984), receptors that recognize NLS-bearing proteins were postulated to exist. Several candidate proteins have been found in both the cytosol and associated with the nucleus (Adam et al

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“…Formation of 29-O-methylated residues is effected by either of two methods. The vast majority of 29-Omethylation of rRNA, and some 29-O-methylation of snRNA, is catalyzed by a common snoRNP complex comprised of Nop1p, Nop58p, Nop56p, and Snu13p subunits (Schimmang et al 1989;Tyc and Steitz 1989;Wu et al 1998;Tollervey 1999, 2000;Watkins et al 2000), which derives its specificity from Box C/D-dependent small nucleolar RNAs (snoRNAs) that guide modification to the appropriate base (Balakin et al 1996;Kiss-Laszlo et al 1996;Yu et al 2005). Alternatively, the formation of 29-O-methylated bases at all sites in eukaryotic and bacterial tRNAs, as well as at some sites in archaeal tRNAs (Clouet-d'Orval et al 2005;Renalier et al 2005), requires specific methyltransferases that act independently of exogenous guide RNAs.…”

Section: Introductionmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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The methylation of the ribose 29-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 29-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 29-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 29-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 29-O-methylation activity, using tRNA Gly(GCC) as a substrate, and extracts made from a trm13-D strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-D strain lack the corresponding 29-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 29-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNA His and tRNA Gly(GCC) with K D values of 85 6 8 and 100 6 14 nM, respectively, and has a K M for tRNA His of 10 nM, but binds nonsubstrate tRNAs very poorly (K D > 1 mM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

Cited by 303 publications

References 60 publications

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

Reconstitution of nuclear protein transport with semi-intact yeast cells.

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

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