Identification and role of cap binding proteins in eukaryotic cells

All eukaryotic cellular mRNAs (except for organelle mRNAs) are blocked at their 5$ sp prime$ end by the cap structure m$ sp7$GpppX (where X = any nucleotide). The cap structure plays a multifunctional role in both the nucleus and the cytoplasm. Two polypeptides of molecular mass of 20 and 115 kDa in HeLa nuclear extracts were shown to recognize and be crosslinked to the cap structure of eukaryotic mRNAs in a cap-dependent fashion. Their cap binding properties have been characterized, although their function in the nucleus is unknown. Previous studies have shown that cap function in the cytoplasm is mediated by eukaryotic initiation factors (eIF)-4A, -4B and -4F (termed cap binding proteins) which interact with the cap structure and are involved in 40S ribosomal subunit binding to mRNA during translation initiation. eIF-4A contains an ATP binding consensus sequence AXXGXGKT, consistent with its ATP binding and hydrolyzing activity. The amino proximal glycine and lysine were mutated to isoleucine and asparagine, respectively, in order to determine the importance of these amino acids in ATP binding as examined by UV-induced crosslinking to ($ alpha sp{32}$P) dATP. Mutation of the lysine, but not the glycine, abolished ($ alpha sp{32}$P) dATP crosslinking to eIF-4A. There has been indirect evidence to suggest that eIF-4A, -4B and -4F interact with the cap structure and use the energy derived from ATP hydrolysis to unwind 5$ sp prime$ proximal secondary structure in mRNA, to facilitate ribosome binding. Using a novel RNA unwinding assay, direct evidence was obtained indicating that combinations of eIF-4A and eIF-4B, or eIF-4F and eIF-4B, exhibit helicase activity. A unique aspect of this activity is that it functions in a bidirectional fashion. The data indicate that cap binding proteins in the cytoplasm, and possibly the nucleus, play a key role in the regulation of gene expression