Identification and Characterization of the Ubiquitously Occurring Nuclear Matrix Protein NMP 238

Box C/D snoRNA-associated proteins: Two pairs of evolutionarily ancient proteins and possible links to replication and transcription

“…Immunoprecipitation of the assembled U14 snoRNP complex with p50 and p55 antibodies+ Antibodies directed against p50, p55, and fibrillarin were used to assess the association of these proteins with U14 snoRNA+ A: Antibodies to p50, p55, and fibrillarin were incubated with the in vitro assembled U14 snoRNP (complex assembled with radiolabeled U14 ⌬AV snoRNA) and then coupled with Protein(A)-Sepharose for subsequent immunoprecipitation+ After extensively washing the Protein(A)-Sepharose beads, bound snoRNP was eluted with 0+1 M glycine, pH 3+0, phenolextracted, and the ethanol-precipitated RNA analyzed on a 10% polyacrylamide-7 M urea gel+ Incubation of the Protein(A)-Sepharose beads with the in vitro-assembled U14 snoRNP complex in the presence (ϩ) or absence (Ϫ) of the designated antibody is indicated+ The marker lane containing radiolabeled U14 ⌬AV is designated+ B: Antibodies to p50, p55, and fibrillarin were incubated with a mouse ascites cell nuclear sonicate and then coupled with Protein(A)-Sepharose for subsequent immunoprecipitation+ After extensively washing the Protein(A)-Sepharose beads, bound snoRNP was eluted with 0+1 M glycine, pH 3+0, and phenol extracted+ The ethanol precipitated RNA was resolved on a 10% polyacrylamide-7 M urea gel and electroblotted to nylon membrane before hybridization with a 32 P-radiolabeled DNA oligonucleotide complementary to the 39-terminal region of mouse U14 snoRNA+ Incubation of the Protein(A)-Sepharose beads with the nuclear sonicate in the presence (ϩ) or absence (Ϫ) of designated antibody is indicated+ The lane designated U14 is a full-length U14 snoRNA marker+ viously reported mouse protein p65 is actually a pair of nucleolar proteins whose homologs in yeast have been designated Nop56p and Nop58p+ Investigations have demonstrated that Nop58 is associated with the box C/D snoRNAs (Wu et al+, 1998;Lafontaine & Tollervey, 1999)+ The binding of mouse Nop58 and mouse Nop56 to the U14 box C/D core motif confirms the interaction of Nop58 with the box C/D core motif and demonstrates that the closely related Nop56 is also a core protein of the box C/D snoRNP complex+ This is consistent with the recent work demonstrating that fibrillarin, Nop58, and Nop56 are three proteins common to all box C/D snoRNAs (Lafontaine & Tollervey, 2000)+ The finding that Nop58 and Nop56 are bound to this RNA motif in vitro attests to the validity of this assembly system for the biochemical/structural study of the box C/D snoRNP core complex+ Previously defined proteins p55 and p50 represent a second, novel pair of highly related nucleoplasmic box C/D snoRNA-associated proteins that have been recently reported with varying nomenclatures (Kanemaki et al+, 1997;Bauer et al+, 1998;Holzmann et al+, 1998;Qiu et al+, 1998)+ During the course of our studies, yeast NOP56 and NOP58 were identified through a genetic analysis as interacting with fibrillarin/Nop1p, a well conserved, box C/D snoRNP protein (Gautier et al+, 1997)+ Immunoprecipitation analysis has since shown that yeast Nop58p is associated with all yeast box C/D snoRNAs (Wu et al+, 1998) as well as Nop58 with vertebrate box C/D snoRNAs (Lyman et al+, 1999)+ Both Nop56p and Nop58p are essential for yeast viability, consistent with a role for these two proteins in box C/D snoRNA biogenesis and stability+ Overexpression of either Nop56p or Nop58p in yeast does not compensate...…”

Pontin is localized in nucleolar fibrillar centers

“…Despite a large number of reports analyzing Pontin functions, a relatively few groups have analyzed Pontin cellular distribution (Bauer et al 1998;Holzmann et al 1998;Makino et al 1998;Salzer et al 1999). However, outcomes of these studies have been inconsistent.…”

“…Pontin localization has been elusive and different groups referred to diverse localization based on the antibody and/or fixation conditions used (Bauer et al 1998;Holzmann et al 1998;Makino et al 1998;Salzer et al 1999). Moreover, a GFP-tag on either end of the protein prevents Pontin localization to the nucleus (data not shown) and thus impairs studying the intranuclear distribution.…”

TIP49b, a Regulator of Activating Transcription Factor 2 Response to Stress and DNA Damage

“…TIP49b (35) (synonyms: TIP48 [73], Reptin52 [3], RUVBL2 [58], Rvb2 [65)], TAP54␤ [31], and TIH2p [24]) is a protein of 463 amino acids that exhibits 43% identity with TIP49a (36) (synonyms: Pontin52 [4)], NMP238 [29)], RUVBL1 [58], Rvb1 [65)], TAP54␣ [31], and TIH1p [40]). …”