Identification of Nopp140-associated proteins, including the novel, conserved NAP65. (A) Immunoprecipitates were separated by SDS-PAGE and visualized by silver staining. Nopp140 was precipitated from rat liver nuclear extracts (lane 1) with peptide antibodies in the absence (lane 2) and presence (lane 3) of competing peptide. The identified polypeptides (closed dots) are listed on the right. The top two dots correspond to Nopp140, as confirmed by immunoreactivity. Open dots indicate unidentified protein bands that specifically coprecipitate with Nopp140. The amino-terminal protein sequences, in single-letter amino acid code, that served to identify NAP65 and fibrillarin are shown in boxes to the right. The circled P above the serine residue in the fibrillarin sequence indicates that it was identified as a dehydroserine, suggesting that this serine was phosphorylated. Xs refer to unidentified residues, all of which correspond to N^G,N^G-dimethylarginines in fibrillarin. The asterisks indicate bands that are not competed for by free peptide representing the Nopp140 IgGs. (B) In vitro transcription/translation of the assembled NAP65 cDNA (pTM136) in the presence of [³⁵S]methionine and [³⁵S]cysteine that was analyzed by SDS-PAGE and fluorography (lane 2). Control reactions contained either no DNA (lane 1) or a plasmid encoding NAP57 (pTM575; lane 3). (C) Protein sequence alignment of NAP65 to the closest homologues of other species. The following lists the species and the sequence sources (EST or GenBank accession numbers) from which the sequences were derived: rat, Rattus species (EST105839 and EST108064); mouse, Mus musculus (AA065550 and AF053232); human, Homo sapiens (AF123534); fly, Drosophila melanogaster (AC004277; 60 amino acids were omitted at position 423 and a frame shift was inserted to align this to the other sequences); worm, Caenorhabditis elegans (AF043704); rice, Oryza sativa (AB015431); pea, Pisum sativum (AF061962); and yeast, Saccharomyces cerevisiae Nop5/58p (AF056070).

NAP57 is a specific component of box H/ACA snoRNPs, whereas Nopp140 associates with both box H/ACA and box C/D snoRNPs. (A) Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by amido black staining. Nopp140 (lanes 2 and 3) and NAP57 (lanes 4 and 5) were precipitated from rat liver nuclear extracts (lane 1) with peptide antibodies in the absence (lanes 2 and 4) and presence (lanes 3 and 5) of competing peptide. Proteins that precipitated exclusively in the absence of competing peptide are listed on the right. NAP65 is indicated by a dot because it stains very poorly by amido black. (B) The membrane in A was probed consecutively with antibodies specific for the antigens listed on the left, and the antibody reactivity was detected by ECL. All of the films showing the ECL results of the individual antibodies were overlaid and are displayed simultaneously. (C) Box H/ACA (top two panels) and box C/D (bottom two panels) snoRNAs were immunoprecipitated from whole cell extracts (lane 1), and their RT-PCR products were separated on agarose gels and detected by ethidium bromide. NAP57 (lanes 2 and 3) and Nopp140 (lanes 4 and 5) were precipitated in the absence (lanes 2 and 4) and presence (lanes 3 and 5) of competing peptide, total RNAs were extracted, and the snoRNAs were amplified by RT-PCR with primers specific for the snoRNAs indicated on the left. To improve the visibility of the precipitated box C/D snoRNA RT-PCR products, the contrast of lanes 2–5 of the bottom two panels was selectively increased.

Rat NAP57 partially complements the function of yeast CBF5. (A) A wild-type yeast CBF5 strain (lane 1) and a conditional strain with CBF5 under the conditional GAL10 promoter (GAL::cbf5; lanes 2 and 3) were grown in permissive medium followed by spotting at the dilutions indicated (dil.) on a restrictive, glucose-containing plate. The strains carried plasmids expressing the indicated proteins. The colonies were imaged after 2 d of growth at 30°C. (B) The same strains shown in A were grown for 24 h in liquid glucose-containing medium, and total RNAs were prepared at 0 h (lanes 1, 3, and 5) and 24 h (lanes 2, 4, and 6). The RNAs were separated on a standard 8% acrylamide gel, transferred to a nylon membrane, and detected by Northern hybridization with ³²P-labeled primers specific for the indicated snoRNAs. (C) A wild-type CBF5 strain (lane 1) and strains with a temperature-sensitive mutation in CBF5 (cbf5^ts; lanes 2 and 3) carrying plasmids expressing the indicated proteins were spotted on a plate as in A. The colonies were imaged after 2 d of growth at 38°C, the nonpermissive temperature.

Transient expression of the dominant negative Nopp140 carboxyl terminus chases snoRNPs out of the nucleolus (A) and arrests RNA pol I transcription (B) without affecting the localization of RNA pol I (C). Cells were transfected with the HA-tagged carboxyl terminus of Nopp140, and subsequently endogenous GAR1 (a) and RNA pol I (c) were labeled by indirect double immunofluorescence or run-on transcription was detected by BrUTP incorporation and visualization with anti-BrdU antibodies (b). The transfected cells (solid arrows) were identified with anti-HA antibodies (a′ and c′) or by the aberrant nucleoplasmic distribution of endogenous Nopp140 detected with Nopp140-specifc antibodies (b′). All nucleoli can be clearly identified in the phase-contrast images (a′′, b′′, and c′′). One nucleolus of a transfected and an untransfected cell is marked in each panel (small open arrows). Bar, 10 μm.

A genetic interaction between Srp40p, the yeast Nopp140 homologue, and box H/ACA snoRNAs. (A) Wild-type yeast (wt), a strain disrupted in SRP40 and in the synthetically lethal gene LES2 but carrying SRP40 on a multicopy plasmid under the control of the conditional GAL10 promoter (srp40Δ les2 [pGAL-SRP40]), and the singly disrupted strains srp40Δ and les2 were spotted at various dilutions on glucose-containing plates (bottom panel) or on plates containing instead raffinose, galactose, and sucrose (top panel). (B) Northern blot of total RNAs extracted from the various strains described in A and indicated on top after growth for 0 and 24 h in glucose-containing medium (odd and even lanes, respectively). The blot was probed consecutively with ³²P-labeled oligonucleotides specific for the snoRNAs listed on the right. The respective class of the snoRNAs is indicated.

Scheme of Nopp140 interactions summarizing our present and previous results and incorporating data from studies in yeast (see INTRODUCTION). The names of the mammalian proteins are printed in bold, and those of the homologous yeast genes are printed in italic. Box H/ACA and box C/D snoRNPs contain not only a separate class of snoRNAs (represented by their conserved secondary structures) but also a distinct set of proteins. Uniquely, Nopp140 interacts with both, although it exhibits a stronger affinity for box H/ACA than for box C/D snoRNPs, as illustrated by the different thickness of the arrows. In addition, Nopp140 appears to be constantly accompanied by small amounts of casein kinase 2 (see also Li et al., 1997) and to interact with the coiled-body protein p80 coilin (Isaac et al., 1998).