Recent findings have revealed that in human cells the C-protein tetramer of nuclear hnRNP complexes binds pre-mRNA in a highly cooperative manner, and that contiguous groupings of three tetramers undergo a combinatorial event ot fold 700 nt lengths of RNA into a unique 19S triad structure. This structure directs the stoichiometric binding of the A and B proteins during 40S core assembly. In the absence of C-protein, the basic A/B hnRNPs bind pre-mRNA to form artifactual aggregates. The presence of one triangular structure in native monoparticles demonstrates its physiological relevance and indicates that C protein is probably the first protein to bind RNA during transcription, and that it functions as a chaperonin to constrain nascent transcript in a state accessible by trans- acting factors. The recent discovery that the tetramer is a dimer of dimers formed by two leucine zipper domains, and that C-protein's high- affinity RNA binding motif is not its RRMs but rather a serine-rich basic motif preceding the zippers, has opened the door to meaningful studies i C-protein's function in RNA processing. In these studies, a yeast expression assay will be used to test the physiological relevance of mutations that effect C-protein's in vitro RNA binding affinities, cooperativity, and RNA folding activity. the same system will be used to screen human genomic libraries for factors that modulate C-protein function in vivo. in a second line of investigation, competition equilibrium binding assays using synthetic and native snRNA and snRNP complexes will be performed to identify RRM binding specificities. Emphasis will focus on the role that snRNA-RRM interactions may play in site-specific displacement of C-protein pre-mRNA and the pleiotropic dissociation of 40S core particles. In a third line of investigation, emphasis will focus on the mechanisms of C-proteins's RNA chaperonin activity. Genetic, biochemical, and multidimensional NMR studies will identify the orientation of the four high-affinity binding sites about the coiled-coil domains, the steric relationship between the RRMs and the high-affinity binding sites, and the elements of primary structure that are critically involved in C-proteins unique RNA binding activities. Finally, in vitro reconstitution assays will be used to identify sites in the tetramer that direct cooperative binding, RNA folding, and stoichiometric core particle assembly. Because the early events of RNA processing occur in the context of a C-protein folded substrate, these studies exist as a prerequisite to understanding the details of mRNA production.
| Rech, J E; LeStourgeon, W M; Flicker, P F (1995) Ultrastructural morphology of the hnRNP C protein tetramer. J Struct Biol 114:77-83 |
| Huang, M; Rech, J E; Northington, S J et al. (1994) The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles. Mol Cell Biol 14:518-33 |
