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The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (13eyer, A. L., M. E. Christensen, 13. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(132). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1 (C2).When isolated nuclei from mammalian cells are disrupted briefly with ultrasound (1-3) or extracted with low ionic strength buffers (4, 5), the majority of the premessenger RNA sequences (heterogeneous nuclear [hn] RNA) ~ are recovered Abbreviations used in this paper. CuP, (orthophenanthrolinehCu(II) complex; DTBP, 3,3'-dimethyl dithiobispropionimidate dihydrochloride; hnRNA, heterogeneous nuclear RNA; hnRNP, heteroge-1570 in 20-25-nm 40S ribonucleoprotein complexes or monoparticles (1-13). Along with hnRNA, these monoparticles are primarily composed of multiple copies of six nucleus-specific polypeptides that migrate in SDS polyacrylamide gels as three groups of doublet bands (A1,A2; B1,B2, and C1,C2) with neous nuclear ribonucleoprotein; NRS, nuclease-resistant structure; OD, optical ...

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins Al and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.The biochemical events of pre-mRNA processing are known largely through studies on the RNA intermediates produced during splicing and studies on the nucleotide sequences required for RNA maturation (for reviews, see references 17 and 42). Nascent transcripts, however, are packaged by a specific subset of major nuclear proteins during transcription to form a ribonucleoprotein (RNP) complex in which the events of RNA splicing occur (see references 8 and 10 for reviews). Historically, these complexes were termed heterogeneous nuclear ribonucleoprotein particles (hnRNP) because the packaged moiety was heterogeneous nuclear RNA and not because the individual particles were known to be heterogeneous in composition and structure (10,38,41). Several observations indicate that nascent transcripts are in fact packaged into a regular repeating structure composed of a contiguous array of 40S hnRNP complexes. These observations suggest that the complexes may play only a passive role in RNA splicing. Other observations suggest that packaging may be transcript specific and thus imply that hnRNP may play a more direct role in the events of RNA processing.Observations in support of a fundamental packaging function for hnRNP are the following. (i) The intranuclear concentration of the major hnRNP is high. For example, in actively growing mammalian cells there is 20 to 30% as much individual core particle hnRNP as individual core particle histone (...

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