General organization of protein in HeLa 40S nuclear ribonucleoprotein particles.

Abstract

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 ...

Autoantibodies to the core proteins of hnRNPs

“…This suggests that the antigenic determinant(s) is (are) not shared between these two groups of proteins, in accord with the peptide mapping results [22]. Interestingly, the core proteins reacting with serum 64b are those thought as being positioned internally to Cr, CZ and A1 [31]. This poses an interesting question as regards the accessibility of the epitopes for the generation of the immune response.…”

Heterogeneous Nuclear Ribonucleoproteins C1 and C2 Associate with the RNA Component of Human Telomerase

“…hnRNPs A1, A2-B1, D, and E have been reported to specifically bind to human single-stranded telomeric repeats (34,44). The C proteins (C1 and C2) are two of the core components of the 40S-hnRNP particle that tether hnRNPs of the 40S particle to hnRNA (41). The C proteins influence pre-mRNA splicing (12), associate with A/U-rich elements that are involved in regulated mRNA turnover (25), and bind downstream of some (61,62).…”

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

“…servations (Samarina et al 1968;Martin et al 1974Martin et al , 1978Pederson 1974;Beyer et al 1977;Karn et al 1977;Maundrell and Scherrer 1979;Walker et al 1980;Le Stourgeon et al 1981;Steitz and Kamen 1981;Lothstein et al 1985;Wilk et al 1985), hnRNA sediments in sucrose gradients as heterogeneous material of greater than 30S and, after mild digestion with nuclease, as more homogeneous material corresponding to monoparticles at about 30S. Heparin converts the hnRNP particles to slightly slower sedimenting material, but it is evident that particles sedimenting only slightly slower than the control remain.…”