Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Abstract

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

“…The slides were fixed for 10 min at room temperature in 4% formaldehyde in 0+1 M PBS (pH 7+4)+ Then, they were permeabilized for 10 min in 100% methanol, rehydrated, rinsed in PBS, and further rinsed in PBS containing 1% BSA (w/v) and normal goat serum (NGS) diluted 1/30+ The cells were placed for 30 min at 37 8C with a monoclonal anti-BrdU antibody (Roche Diagnostics) diluted 1/100 in PBS, containing NGS diluted 1/50 and 0+2% BSA+ After rinsing with PBS containing 1% BSA, the slides were incubated for 30 min at 37 8C with FITC-conjugated goat anti-mouse antibody (Sigma) diluted 1/100 in PBS containing 0+2% BSA+ After several rinses, the slides were mounted with Citifluor TM AF1 (Agar Scientific, Starsted, United Kingdom)+ For double labeling experiments, the slides were fixed for 5 min at room temperature in 4% formaldehyde in 0+1 M PBS (pH 7+4) containing 1% Triton X100+ They were rinsed first in PBS, then in PBS containing 1% BSA, as well as NGS and normal sheep serum (NSS), both diluted 1/30+ They were incubated for 30 min at 37 8C with an anti-BrdU antibody and a rabbit polyclonal antibody against RNA polymerase I (a gift from Dr+ K+ Rose, Houston, Texas) or a human autoantibody to fibrillarin (S4; Ochs et al+, 1985), both diluted 1/100 in 0+1 M PBS containing 0+2% BSA, NGS, and NSS, both diluted 1/50+ The slides were washed with PBS containing 1% BSA and incubated for 30 min at 37 8C with an FITCconjugated goat anti-mouse antibody (Sigma, for BrUTP) and a biotinylated sheep anti-rabbit antibody (Roche Diagnostics, for RNA Polymerase I) diluted 1/100 in PBS containing 0+2% BSA+ This secondary antibody was detected with streptavidin labeled with Texas Red (Amersham Life Science, Little Chalfont, United Kingdom) diluted 1/100 in PBS containing 0+2% BSA for 10 min+ After rinsing, the cells were mounted with Citifluor TM AF1+…”

Clinical and Prognostic Associations Based on Serum Antinuclear Antibodies in Japanese Patients with Systemic Sclerosis

“…The followup period was <5 years in 85 patients, [5][6][7][8][9][10] years in 53 patients, and > 10 years in 75 patients. During the period of observation, 99 patients received corticosteroids (mean maximum dosage 33 mg of prednisone equivalent daily), 23 received D-penicillamine, and 16 received cytotoxic drugs (11 took azathioprine, 3 methotrexate, and 2 mizoribine).…”

U17XS8, a small nucleolar RNA with a 12 nt complementarity to 18S rRNA and coded by a sequence repeated in the six introns ofXenopus laevisribosomal protein S8 gene

“…Partial characterization of U17xS8 containing RNP has revealed that 1) the monoparticle sediments at about 12S, sligthly faster than the U3 RNP, while a bigger complex sediments at more than 40S as it occurs also for other snoRNPs; 2) it has a density of 1.45 g/ml, which implies that the particle contains about 30% of RNA and 70% of protein; 3) it is not precipitated by antibodies against Xenopus fibrillarin (27), as expected by the absence of the C and D boxes typical of fibrillarin associated RNAs (28).…”