Nuclear RNA Fractions Obtained by Sequential Extraction of Rat Liver Nuclei with Salt Solutions and their Relationship to Polysome‐Associated RNA

Abstract

Pulse-labeled RNA from rat liver nuclei was fractionated by sequential treatment of purified nuclei with buffered salt solutions of Werent ionic strength in the presence of ribonuclease inhibitor prepared from rat liver cytoplasm. Two fractions of pulse-labeled nuclear RNA were successively extracted from rat liver nuclei with 0.14 M NaC1, 0.001 M MgCl, a t pH 7.0 and pH 8.0, respectively. The labeled material isolated a t pH 7.0 sediments between 6-16 S while the pulse-labeled RNA extracted a t pH 8.0 sediments in the range of 10-34 S. Both fractions are characterized by a high content in AMP. With respect to [3aP]nucleotide composition, sedimentation characteristics and hybridizability, these fractions correspond closely to non-ribosomal RNA associated with polysomes. When a third extraction is carried out a t pH 8.0 in 0.3 M NaCl another rapidly labeled RNA component is obtained. This RNA sediments above 34 S, turns over more rapidly than the RNA species extracted in 0.14 M NaC1, and contains less AMP. As judged by [32P]base-composition, sedimentation and hybridizability, this RNA component is not present in polysomes. The pulse-labeled RNA remaining in the nuclei after three salt extractions comprises mainly ribosomal or ribosomal precursor RNA as well as DNA-like RNA species which show little or no nucleotide sequence homology with polysome-associated RNA.Since nuclear RNA species clearly different from polysome-associated cytoplasmic RNA are released from nuclei only at a relatively high ionic strength and are retained a t a physiologic salt concentration it is suggested that RNA species not designated to function in cytoplasmic protein synthesis are a priori bound more tightly to nuclear structures than those types of RNA which serve as vehicles for the nucleo-cytoplasmic transfer of genetic information.Recent reports from a number of laboratories have indicated that only a fraction of non-ribosomal RNA synthesized in the nuclei of mammalian cells is eventually released into the cytoplasm [l-41. A substantial portion of newly-formed DNA-like RNA appears to be subjected to a rapid turnover in the nucleus itself [5,6]. The experimental evidence which gave rise to such concepts is indirect: it was shown that the kinetics of labeling of DNA-like RNA from nuclei, nucleoplasm, cytoplasm and from purified polysomes were clearly different and virtually excluded a simple precursor product relationship between these RNA species [l, 5,7]. Moreover, hybridization-competition experiments demonstrated that cytoplasmic or microsomal RNA from various mammalian cell species can compete with the respective labeled nuclear RNA for DNA binding sites only to a very limited degree [2,3,8].A higher degree of nucleotide sequence homology between nuclear RNA and microsomal RNA than in normal liver was observed in regenerating liver [8,9]. 18.I n a number of Morris hepatomas this homology appeared to be nearly complete [8]. On the basis of these results, it was postulated that the stringency of the selection mechanism operating at...

HnRNP particles

“…Hybridization experiments between RNA or DNA complementary to mRNA and purified hnRNA (15,38,40,(43)(44)(45)(46)(47) indicate that mRNA sequences are found in hnRNA molecules. Further evidence on this point can be found in the successful translation of hnRNA in oocytes and cell-free systems (3,(48)(49)(50), from kinetic studies with and without transcriptional inhibitors (8,38) and from the similarity in the types of posttranscriptional modification characteristic of hnRNA and mRNA (38).…”

“…The presence of poly(A) and oligo(A) sequences The 3'poly(A) segments characteristic of most cytoplasmic mRNAs have been shown to be present in hnRNA and hnRNP complexes (11,17,18,39,43,58). The poly(A) is added posttranscriptionally by a terminal addition enzyme (see for example 59).…”