Distribution of snRNP complexes in rat liver nuclear extracts: biochemical and immunochemical analysis

Abstract

Rat liver nuclei were extracted with 0.14 M NaCl and the extracts submitted to sucrose gradient fractionation. Aliquots of the nuclear residue remaining after the 0.14 M NaCl extraction were also extracted either with 0.3 M NaCl or 1 M urea, and the extracts similarly submitted to sucrose gradient fractionation. Thereafter, both the presence and relative distribution of individual U-snRNA (U1-U6) species was followed. Results showed an extensive association of all U-snRNAs to RNP structures of greater than or equal to 40 S. However, characteristic differences in the association of mostly U1 and U5--which were the major identifiable species in the extracts--to these structures were observed. Only a small fraction of U1 appeared complexed to less than or equal to 40 S RNP structures, while most of it sedimenting in the greater than 20 S region of the gradient. In contrast, U5-snRNA had a tight and almost exclusive association to 40 S RNP structures. No pool of 10-12 S U5-snRNP complexes was detected. Combined immunoprecipitation and immunoblotting experiments on nuclear 0.14 M NaCl extracts using anti-Sm and/or anti-RNP antisera showed that all snRNA species, whether recovered as 10-12 S complexes or segregated with greater than or equal to 40 S RNP components, existed as snRNP structures bearing at least their Sm-antigenic polypeptides. These and our previous results [Guialis, A., Arvanitopoulou, A, Patrinou-Georgoula, M. and Sekeris, C.E. (1983) FEBS Lett. 151, 127-133], support the existence of snRNP-enriched RNP structures of greater than or equal to 40 S. In such structures the core polypeptides (Mr = 32,000-45,000) of 40 S monoparticles are not obligatory components.