THE RIBONUCLEASE-ACTIVITY OF NUCLEOLAR PROTEIN B23

Protein B23 is an abundant nucleolar protein and putative ribosome ass embly factor, The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three pur ified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein w ere found to have ribonuclease activity. However, the specific activit y of recombinant B23.1 was similar to 5-fold greater than that of reco mbinant B23.2. The activity was insensitive to human placental ribonuc lease inhibitor, but was inhibited by calf thymus DNA in a dose depend ent manner. The enzyme exhibited activity over a broad range of pH wit h an apparent optimum at pH 7.5. The activity was stimulated by but no t dependent on the presence of low concentrations of Ca2+, Mg2+ or NaC l. The Ca2+ effect was saturable and only stimulatory in nature. In co ntrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these opt ima, These data suggest that protein B23 has intrinsic ribonuclease ac tivity, The location of protein B23 in subcompartments of the nucleolu s that contain preribosomal RNA suggests that its ribonuclease activit y plays a role in the processing of preribosomal RNA.