Proteins of rat liver nucleolar RNP, especially 110S RNP containing 45S RNA, were investigated and the following results were obtained.
1. Nucleolar extract was prepared from thioacetamide-treated rat liver nucleoli in the presence of 1mg PVS per ml. Sucrose-density gradient centrifugation of the nucleolar extract showed RNP distributed between 60S and 1105. The 110S particles (110S RNP) contain 45S RNA as judged from the radioactivity profiles of nucleolar extract labelled in vivo with [³H] orotic acid for 10min and 2 h. The protein components of 110S RNP and total RNP heavier than 60S (total RNP) were analyzed and compared.
2. Since the effects of PVS treatment on the extraction and the electrophoretic pattern of ribosomal proteins were removed by increasing the Mg²⁺ concentration during acetic acid extraction, as described in our preceding paper (1), proteins of 110S RNP as well as total RNP from PVS-pretreated nucleoli were extracted with 67% acetic acid containing 334 mM Mg²⁺ and analyzed by two-dimensional acrylamide gel electrophoresis. Ribosomal proteins masked by contaminating histone spots on the gel were identified by SDS-acrylamide gel electrophoresis.
3. 110S RNP contained a large portion of ribosomal proteins from large subunits; 24 protein spots were distinct, 6 protein spots were faint and 5 proteins (L3, L8, L30, L35, and L36) were missing completely. On the other hand, 110S RNP contained a small number of small subunit proteins; only 6 proteins showed distinct spots, 7 proteins showed faint spots and 12 protein spots were missing. 110S particles contained 11 non-ribosomal proteins, although it is difficult to rule out the possibility that some of them were contaminating chromatin proteins.
4. Total RNP showed electrophoretic patterns of 60S ribosomal proteins similar to those of 110S RNP, although L3 protein was present as a faint spot and 26 proteins showed distinct spots. Total RNP contained more small subunit proteins than 110S RNP; 7 proteins spots were distinct, 10 protein spots were faint and 8 protein spots were missing. The results suggest that some kinds of 40S proteins and L3 protein are attached to 110S RNP during the processing of 110S RNP.