James McAfee

Biochemistry Research Summary

In eukaryotic cells pre-mRNA is initially complexed with ~ 30 abundant nuclear proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs). Though the function of hnRNPs in RNA metabolism is an area of scientific controversy, there is a consensus of opinion that they play key roles in the biogenesis of mRNA. They have been implicated as performing such diverse roles in RNA metabolism such as pre-mRNA capping, pre-mRNA splicing, polyadenylation and nuclear export. Several of the hnRNPs have been suggested to be involved in the regulation of gene expression as well as regulating the length of chromosomal telomeres. One of the most abundant of the hnRNP proteins is hnRNP C. There are two isoforms of hnRNP C known as C1 and C2. hnRNP C2 differs from C1 by the presence of a 13 amino acid insert that results from alternative splicing of the same pre-mRNA. These two proteins are reported to be involved in the packaging of pre-mRNA as well as RNA splicing. Recently, a protein very similar in primary sequences to hnRNP C1 and C2 known as p542 was recently discovered. The role of this protein in RNA metabolism is not known but its sequence similarity to hnRNP C suggest comparable functions for both proteins. However, targeted disruption of either the hnRNP C or p542 is embryonic lethal, suggesting that the two proteins are not functionally redundant. A primary objective of the biochemistry laboratory is to identify primary sequence elements or motifs in both proteins that generate functional identity or functional redundancy. To accomplish this objective the laboratory has cloned and expressed the human gene for p542 in E. coli. Interestingly, we have found that there exist at least 3 different isoforms of the p542 protein. Our future objectives are to use site specific mutagenesis to generate C-protein-p542 chimeras to assess the role that primary determinants of each protein play in function. Protein function is assessed in the laboratory by evaluating thermodynamic parameters for the interaction of recombinant proteins with RNA using fluorescence spectroscopy. We have also developed an in-vivo assay for determining RNA binding using a yeast expression system.