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Vol. 10, Issue 1, 77-90, January 1999
Department of Cell Biology and Anatomy, Mount Sinai School of
Medicine, New York, New York 10029-6574
rRNA precursors are bound throughout their length by specific
proteins, as the pre-rRNAs emerge from the transcription machinery. The
association of pre-rRNA with proteins as ribonucleoprotein (RNP)
complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and
through assembly of ribosomal subunits in the nucleolus. Preribosomal
RNP complexes contain, in addition to ribosomal proteins, an unknown
number of nonribosomal nucleolar proteins, as well as small nucleolar
RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a
specific, rapid, and mild immunopurification approach to isolate and
analyze human RNP complexes that contain nonribosomal nucleolar
proteins, as well as ribosomal proteins and rRNA. Complexes
immunopurified with antibodies to nucleolin
a major nucleolar
RNA-binding proteincontain several distinct specific polypeptides
that include, in addition to nucleolin, the previously identified
nucleolar proteins B23 and fibrillarin, proteins with electrophoretic
mobilities characteristic of ribosomal proteins including ribosomal
protein S6, and a number of additional unidentified proteins. The
physical association of these proteins with one another is mediated
largely by RNA, in that the complexes dissociate upon digestion with
RNase. Complexes isolated from M-phase cells are similar in protein
composition to those isolated from interphase cell nuclear extracts.
Therefore, the predominant proteins that associate with nucleolin in
interphase remain in RNP complexes during mitosis, despite the
cessation of rRNA synthesis and processing in M-phase. In addition,
precursor rRNA, as well as processed 18S and 28S rRNA and candidate
rRNA processing intermediates, is found associated with the
immunopurified complexes. The characteristics of the rRNP complexes
described here, therefore, indicate that they represent bona fide
precursors of mature cytoplasmic ribosomal subunits.
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