The association of nucleolar
phosphoprotein C23 with preribosomal
ribonucleoprotein (RNP) particles was examined in
Novikoff hepatoma nucleoli.
RNA was labeled with [3H]
uridine for various times in cell
suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by
sucrose gradient ultracentrifugation. The majority of
protein C23 cosedimented with fractions containing rapidly labeled
RNA (RL fraction). To determine whether there was a direct association of
RNA with
protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to
phenol/
chloroform extractions, as much as 30% of the labeled
RNA was found in the
phenol (
protein) layer, indicating that
RNA became cross-linked to
protein. Similarly, there was an increase in
protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than
protein C23, appearing as a smear detected either by [3H]
uridine radioactivity or by anti-C23 antibody. With anti-C23
antibodies, up to 25% of the labeled
RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned
rDNA fragments as probes, indicated that the
RNA in the RL fraction and the immunoprecipitated
RNA contained sequences from 18S and
28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)