A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.Whether released from isolated nuclei by sonic disruption or by low-salt extraction, the majority of the pre-mRNA molecules remain dispersed in solution following chromatin removal by brief centrifugation. Under conditions of minimal nuclease activity, 70 to 95% of this RNA is recovered in large ribonucleoprotein (RNP) complexes which sediment from 30S to more than 200S (26, 39, 52). Electron micrographs of the faster-sedimenting complexes reveal 20-to 25-nm particles arranged either as an array of polyparticles (29,36,42,43,52,53,61) or as clusters of particles when nuclease activity is aggressively inhibited (56). Upon mild nuclease activity the polyparticle complexes are lost and the cleaved RNA (mostly 500-to 1,000-nucleotide [nt] fragments) is recovered in 20-to 25-nm 30S-40S monoparticles (heterogeneous nuclear RNP [hnRNP] particles or ribonucleosomes) (3, 10, 18, 56, 64). Monoparticles purified from HeLa nuclei via glycerol gradients are primarily composed of six abundant nuclear proteins (the core particle proteins) (10, 28, 54, 65), which exist as three heterotypic tetramers, (A1)3B2, (A2)3B1, and (C1)3C2 (4, 7, 39). The (A2)3B1 and (C1)3C2 tetramers have been isolated and partially characterized (4, 7). The (A1)3B2 tetramer has not been isolated, but its existence is inferred from chemical cross-linking studies which reveal that Al exists in monoparticles as homotrimers (34, 41) and in a 3:1 ratio with B2. Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50) (11,17,35,38,46,65).Isolated 40S monoparticles completely dissociate upon RNA digestion with nuclease (22, 64). Spontaneous reassembly occurs in vitro upon the addition of 700 ± 20 nt of exogenous RNA or single-stranded DNA (22). Multiples of this length support the spontaneous in vitro assembly of dimers, trimers, and polyparticle complexes (22, 39). Reconstituted particles possess the same sedimentation coefficient, protein stoichiometry, chemical and UV cross-linking properties, pattern of salt-induced protein dissociation, nuclease sensitivity, and ultrastructural morphology as native hnRNP (22,27,64). Most of the noncore proteins present in initial hnRNP preparations do not quantitatively reconstitute with the core particle proteins (22, 64), a finding which indicat...

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A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.

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