Heterogeneous nuclear ribonucleoprotein C (hnRNP C) is ubiquitous in vertebrates, and exists as two alternatively spliced isoforms, hnRNP C1 and hnRNP C2 (hnRNP C).  hRaly and hRalyl have been added to the C protein family based on primary sequence similarity with hnRNP C1 and C2.  In addition, hRaly and hRalyl conserve functional domains that have been identified for hnRNP C including an amino terminal RNA binding domain (RBD), a basic region that precedes a leucine zipper (bZIP motif), and an acidic carboxy terminus.  The 28 amino acid leucine zipper has been shown to mediate protein-protein interactions between hnRNP C protomers through the formation of a coiled-coiled 4-helix bundle.   The primary sequence conservation of this region in hRaly and hRalyl protomers, as well as its positional conservation raises the possibility of heterotypic interactions between hnRNP C, hRaly, and hRalyl polypeptides. To address this possibility, the cDNA sequence of hRaly and hRalyl were cloned into the polycistronic vector, pST44 to produce pST44/hRaly/hRalyl.  However, pST44/hRaly/hRalyl could not be established in Escherichia coli strain BL21 DE3 RIPL but was stable in E. coli BL21 DE3 pLysS.  Furthermore, plasmid constructs containing either hRaly or hRalyl alone could be propagated in both E. coli expression strains. We propose that lethality could result from leaky expression of the pST44/hRaly/hRalyl construct or through a functional change in the protein as a result of the formation of hetero-oligomers.

Analysis of heterotypic protein-protein interactions between hRALY and hRalyl protomers

247th National Meeting (2014)

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