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.

The PPGMRPP repetitive epitope of the Sm autoantigen: Antigenic specificity induced by conformational changes. Application of the Sequential Oligopeptide Carriers (SOCs)

“…Sera from patients with autoimmune connective tissue diseases, namely, systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD) and primary Sjogren's syndrome (pSS) were tested (i) for anti-Sm and anti-U1RNP reactivity with URNAs precipitation [20] and (ii) for anti-Ro(SSA) and anti-La(SSB) reactivity with counterimmunoelectrophoresis [21 ].…”

Changes in UsnRNA biosynthesis during rat liver regeneration

“…Identification of UsnRNA bands on the scanning profile were accomplished by comparing their mobilites with that of known cytoplasmic marker RNAs namely 4S, 4.5S, 5S and 5.8S, as well as by direct comparison to snRNAs isolated from whole nuclei [30,31]. Quantitation by densitometry after silver staining was done under the condition that has given a linear response with the RNA concentration [29].…”

Autoantibodies to the core proteins of hnRNPs

“…Monoparticles segregate within the 40-50 S range of the gradient (region 5 in fig.2A), lo-12 S snRNP particles are recovered from the light regions of the gradient (regions 2 and 3), while heavier than 60 S RNP structures can be found in the 50% sucrose cushion (region 6) of the gradient [20].…”