Interaction of hnRNP-C1/C2 proteins with RNA: analysis using the yeast three-hybrid system

Three-hybrid assays for the analysis of RNA-protein interactions in vivo are usually used, due to technical limitations, only for RNA baits that do not contain runs of four or more consecutive uridines. The present study provides the first example of a three-hybrid analysis of synthetic and natural uridine-rich RNA sequences. The use of the three-hybrid assay enabled us to demonstrate a functional difference between two closely related proteins, heterogeneous nuclear ribonucleoprotein C1 (hnRNP-C1) and hnRNP-C2. The hnRNP-C2 protein, an alternatively spliced variant of hnRNP-C1, contains an additional 13 amino acids between an RNA binding domain (RBD) and a basic leucine zipper-like motif (bZLM), also implied in RNA binding. This study shows that (i) for efficient binding of hnRNP-C1/C2 to RNA, the context of the U-stretch is more important than the stretch itself; (ii) both the RBD and the bZLM bind RNA independently; and (iii) the C2-related 13-amino acid insert enhances the specificity of either the RBD, the bZLM, or the full-length protein towards its ligand, allowing it to bind only the most high-affinity sequences while discriminating against those that do not perfectly match this category. The three-hybrid system is a powerful tool to work out the functional significance of peptide 'modules' within RNA binding proteins generated by alternative splicing.