An ultrastructural characterization of thehnRNP C protein tetramer and in vitro C protein-RNA assembly intermediate

Extensive biochemical and ultrastructural evidence indicates that heterogeneous nuclear ribonucleoproteins (hnRNP) package pre-mRNA molecules into regular, repeating ribonucleoprotein complexes or 40S hnRNP particles. The six core hnRNP found in association with pre-mRNA: A1, A2, B1, B2, C1, and C2 are isolated in the stoichiometric ratio of 3:3:1:1:3:1. Electron microscopy of the purified C protein tetramer ((C1)$sb3$C2) reveals four-lobed structures with an average diameter of 85A in negative staining. These results confirm the tetrameric arrangement of polypeptides previously suggested. In good agreement with negative staining, a tetramer diameter of 92A was determined by rotary shadowing techniques. Ultrastructural and biochemical research indicates that three C protein tetramers associate with particle-length or 700 nts of RNA to form discrete triangular structures (19S complexes) with 230A sides and a 180A base. Further experiments confirm that each tetramer associates with approximately 230 nts of RNA. These results provide the first explanation for the length-dependent packaging phenomenon demonstrated by hnRNP proteins and the 700nt RNA requirement for monoparticle formation. These findings suggest that C protein tetramers bind all along nascent transcripts, presumably independent of sequence. The intrinsic ability of the C protein tetramer to package RNA into regular and repeating RNP complexes was demonstrated by the formation of two triangle complexes on 1400 nts of RNA and three-triangle complexes on 2100 nts of RNA. Furthermore, this triangular-shaped C protein-RNA complex was recovered from native and reconstituted 40S hnRNP monoparticles after salt dissociation of the A and B proteins. These C protein-RNA complexes were also recovered after UV crosslinking and high salt dissociation of monoparticles. These results suggest a relationship between the 19S complex and the association between C proteins and RNA in monoparticles. In vitro biochemical assembly experiments demonstrate that only the 19S complex can support the addition of the other core hnRNP in the formation of stoichiometric 40S hnRNP monoparticles. Both ultrastructural and biochemical results suggest that the C protein tetramer is responsible for organizing the assembly of hnRNP into regular, repeating complexes in the in vitro formation hnRNP monoparticles.