Abstract
Heterogeneous nuclear RNA (HnRNA) is associated with a set of specialized RNA-binding proteins. Mild ribonuclease digestion of intact HnRNA-protein complexes released 15 S ribonucleoprotein (RNP) particles containing poly(A) and its associated protein and 40 S RNP particles containing most of the HnRNA sequences. Highly purified 40 S RNP particles have been obtained from rat liver by centrifugation of nuclear extracts on sucrose density gradients and isopycnic banding on Metrizamide density gradients. These RNP preparations contain 27% of the total HnRNA sequences of rat liver, and appear homogeneous when viewed in negative contrast in the electron microscope and by centrifugation studies using velocity sedimentation in sucrose density gradients or isopyenic banding in density gradients of cesium salts. Analysis of the proteins in the rat liver 40 S RNP particles by two-dimensional gel electrophoresis demonstrated that the 40 S RNP particle is composed of 12 major protein components with molecular weights ranging from 29,000 to 42,000 which accounted for 75% of the total protein mass and 13 minor protein components with molecular weights greater than 42,000. The proteins in the 29,000 to 42,000 group were fractionated by ion exchange chromatography. The amino acid compositions of the purified protein fractions were strikingly similar and shared several unusual features that distinguish these HnRNA-associated proteins, as a group, from the histones and the non-hi&one chromosomal proteins. Each of the RNP proteins have basic charge characteristics (p1 greater than 8.0) high glycine (25 mol %), low cysteine, and little detectable methionine. Like the histones, the HnRNP proteins are subject to extensive postsynthetic modification. We have identified the unusual amino acid NC, NC (CH,),-L-arginine in acid hydrolysates of some of the RNP proteins and shown that this amino acid
Highlights
RnM MgCl,) to remove sucrose and aliquots were treated for 30 min at 0” with 0.1 pg/ml of pancreatic ribonuclease (Worthington) to generate 40 S RNP particles which were purified on linear 15 to 30%
The distribution of RNA, protein, and DNA at successive stages in the fractionation of hepatocyte nuclei is shown in RNA remaining in the insoluble fraction is associated with nucleoli and probably represents ribosomal RNA sequences, we conclude that the pH 8.0 extract contains over 80% of the newly synthesized nonribosomal. This estimate is in close agreement with the original data of Samarina et al [1] who reported that nearly 80% of the nuclear RNA of DNA-like base composition is extractable from rat liver nuclei in hypotonic buffers
Similarity of Protein Components of 40 S HnRNP Particles from Different Species -To what extent are the organizational principles of rat liver ribonucleoprotein particles we have described applicable to ribonucleoprotein structures in other species? HnRNP particles have been isolated from human (HeLa S-3) cells and from duck liver cells and their protein components compared by electrophoresis in polyacrylamide gradient gels containing 0.1% sodium dodecyl sulfate (Fig. 14)
Summary
The 3’region of HnRNA is released by mild ribonuclease treatment of isolated HnRNP fractions, and sediments tngether with its associated proteins as a particle of about 15 S [8, 10, 14, 17]. The highly purified RNP monomer particles we have obtained appear homogeneous in the electron microscope when viewed in negative contrast and by centrifugation studies using velocity sedimentation in sucrose density gradients or isopycnit banding in density gradients of cesium salts, as well as by agarose gel electrnphoresis following fixation with bifunctional reagents. The proteins in the 29,000 to 42,000 molecular weight group were fractionated by ion exchange chromatography and shown to have closely related charges and amino acid compositions,.
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