Selection of a preribosomal RNA processing site by a nucleolar endoribonuclease involves formation of a stable complex.

Abstract

A nucleolar endoribonuclease from mouse Ehrlich ascites tumor cells, that has been implicated in the endonucleolytic cleavage of mouse precursor ribosomal RNA, specifically and stably binds an in vitro-derived rRNA transcript containing the +650 early processing site. The specificity of binding was demonstrated by mobility shift analysis, glycerol gradient velocity sedimentation analysis, and UV-crosslinking studies. Binding did not require Mg2+ and therefore was not dependent on cleavage; however, binding was dependent on the presence of the early +650 processing site since a pre-rRNA transcript with the +650 processing site deleted failed to compete in binding. A small nucleolar RNA component was not required for the formation of this stable complex or for the specific cleavage of a processing competent pre-rRNA transcript. UV crosslinking studies using 32P-labeled 5-azidouridine-substituted pre-rRNA with bound nucleolar endoribonuclease identified three closely sized polypeptides of approximately 50, approximately 48, and approximately 45 kDa, respectively, that specifically crosslinked to the processing competent rRNA transcript. These three polypeptides species were identified following ribonuclease digestion and electrophoresis on a SDS-polyacrylamide gel. An identical pattern of labeled polypeptides was also identified from gel mobility shift analysis where the specifically shifted material was U.V. crosslinked. The largest of these polypeptides corresponded to the estimated size of the nucleolar endoribonuclease, while the lower molecular weight species may represent partially proteolyzed enzyme. Overall, these results suggest that the unique specificity of the nucleolar endoribonuclease may, in part, be attributed to the formation of a stable complex at the +650 processing site for mouse preribosomal RNA, and that formation of this unique stable complex affords a means to specifically label the limited amount of available partially purified enzyme for sequence analysis.