Cap structures, cap-binding proteins, and their role in regulation of gene expressionAlmost all eukaryotic mRNAs possess at their 5′ ends a cap structure consisting of 7-monomethylguanosine that is connected via a 5′-5′ triphosphate bond to the next nucleoside, which is often 2′ O-methylated (m7GpppN; MMG cap). Uridine-rich small nuclear RNAs (U snRNAs), which play a key role in splicing mRNA precursors, are capped with a 2,2,7-trimethylguanosine (m32,2,7GpppN; TMG cap). About 70% of nematode mRNAs become TMG-capped by the addition of a spliced leader to their pre-mRNA via trans splicing. In unicellular kinetoplastida, the cap is m7Gpppm36,6,2′Apm2′Apm2′Cpm3,2′U, known as cap-4, and all nuclear mRNAs acquire this cap by trans splicing.
The best-studied MMG-cap-binding protein is the cytoplasmic translation initiation factor eIF4E, which is part of a larger complex, called eIF4F, containing the scaffolding protein eIF4G and the ATP-dependent RNA helicase eIF4A. Solved by NMR and X-ray crystallography, the three-dimensional structures of murine, yeast, and human eIF4E show a conserved cap-binding pocket, which consists of a sandwich of two tryptophan residues followed by a region of basic amino acids that ensure favorable interaction with the negatively charged phosphate bridge. Expression of eIF4E is elevated in many oncogenically transformed cell lines and is correlated with malignancy (breast cancer and head, neck, and lung carcinomas). Multiple eIF4E proteins may be found in the same organism; for example, five isoforms were identified in Caenorhabditis elegans. Three recognize the MMG cap exclusively, whereas the other two recognize both the MMG and TMG caps. A nuclear cap-binding complex (CBC), which consists of a cap-binding protein (CBC20) and a homologue of eIF4G (CBC80), has been implicated in nuclear transport, a “pioneer” round of translation, and RNA processing. The crystallographic structure of the free 20/80 CBC complex (apo) and of the complex with m7GpppG shows that two tyrosine residues from the 20-kD subunit interact via stacking with 7-methylguanine.
Cap analogues as tools in the search for biochemical and biophysical mechanisms of gene expression regulationDuring the last two decades, we developed a series of synthetic 5′ RNA cap analogues as valuable tools for studying the molecular mechanisms of gene regulation. The mono- and dinucleotide cap analogues, modified within the base, ribofuranose ring, and phosphate chain, have proven useful in studying the function of eIF4E in cap-dependent translation. Competing for eIF4E with the native mRNA cap, they inhibit in vitro protein translation in rabbit reticulolysates. Long before the crystallographic and NMR complexes of cap-eIF4E were solved, the analogues enabled us to identify the structural parameters within the MMG cap responsible for its interaction with eIF4E. We first synthesized the natural TMG cap (m32,2,7GpppG), which was helpful in deciphering the intracellular transport pathway of U snRNPs. An affinity resin with m32,2,7GTP provided an extremely important tool for the isolation and characterization of five eIF4E isoforms from C.elegans (in collaboration with Robert Rhoads of Louisiana State University) and eIF4E-3 from Ascaris suum (with Richard Davis of the City University of New York). Additional tools that we developed include the anti-reverse cap analogues, which enable the in vitro synthesis of RNA transcripts with higher translational efficiencies than mRNAs capped with natural m7GpppG (with Robert Rhoads). Recently, we synthesized the trypanosomatid cap-4, which has been crucial for characterization of the eIF4E isoforms from Leishmania (in collaboration with Michal Shapira of Ben-Gurion University of the Negev, Beer Sheva, Israel) and should facilitate a variety of future studies on the importance of this unique cap structure in these human parasites.
Fluorescence studies of proteins that specifically bind to mRNA 5′ capIn a close collaboration with Richard Stolarski's group in our department, we developed fluorescence and calorimetry techniques for studying the structural and dynamic aspects of the interactions between the eIF4E factors from various sources (human, murine, yeast, and C.elegans) and synthetic mRNA cap analogues. We applied time-synchronized titration, a new, unambiguous method of fluorescent titration, under various pH and ionic strength conditions, using a long series of cap analogues. The values of Kas calculated for structurally modified cap analogues and the analysis of the eIF4E-cap complexes gave us the Gibbs free energy of binding, the van't-Hoff enthalpy, and the entropy of the protein-cap association. These studies enabled us to interpret structural elements of the cap that are key determinants of the physical interactions, interactions that contribute to the affinity binding of eIF4E proteins. Our investigation of the molecular mechanism that regulates the translation process by eIF4E phosphorylation began with the construction, using an intein-mediated protein ligation strategy, of the protein phosphorylated at Ser209, as well as the protein's mutated forms. Fluorescence titration revealed a surprising change in the cap affinity for the phosphorylated protein compared with its unphosphorylated form; phosphorylation of eIF4E attenuates the factor's interaction with mRNA 5′ cap analogues by electrostatic repulsion.
Spectrofluorometric titrations of the CBC with cap analogues showed a significant difference between in the specificities of CBC and eIF4E toward particular structural elements of cap structure, a finding that may have crucial implications for the biological activities of both proteins.
Synthesis and use of new biologically and medically relevant cap analoguesUnlike their human counterparts, nematode cap-binding proteins can interact with both m32,2,7GpppN and m7GpppN. Thus, they have unique characteristics and represent novel potential targets for rational drug design and the development of nematocides. Parasitic nematodes infect three billion humans, leading to considerable morbidity; they also pose a significant problem for livestock and domestic animals. Trans splicing is also present in other metazoan parasites of human importance, such as schistosomes and tapeworms. Thus, the cap-interacting proteins that have evolved to accommodate the presence of the m32,2,7GpppN cap on mRNAs as a result of trans splicing present a common potential target in all these metazoan parasites. The cap-4 present in all trypanosome mRNAs also presents a unique target. While the murine protein distinguishes between the two cap structures, the parasite protein LeishIF4E-1 binds to both equally well. We believe this unique interaction between cap-4 and the parasite eIF4E could serve as an ideal drug target. Therefore, we plan to examine whether new cap analogues may mimic cap-4 and inhibit translation in a semipermeable cell system. We will also examine whether the cap-4 structure can be replaced in vivo and whether it is dispensable for translation. This work will be done in collaboration with the Shapira lab. We will also use the synthetic cap-4 for NMR studies on the interaction of this structure with the parasite protein, in collaboration with Gerhard Wagner's lab at Harvard Medical School.
Independently, we have been developing an approach to synthesize cap analogues that are able to cross the plasma membrane as in vivo inhibitors of translation (potential anticancer and antiparasitic drugs). We link cap analogues to different lipophilic ligands that can target and inhibit protein synthesis in cancer cells overexpressing eIF4E.
We are designing and synthesizing another series of cap analogues to study the decapping enzymes DcpS (scavengers) and Dcp2 from humans, nematodes, yeast, and trypanosomes. Introduction of methylene or sulfur into the triphosphate bridge leads to protection against decapping activities of DcpS and Dcp2 enzymes. We are in the process of employing a broad library of synthetic cap dinucleotides (those currently available and ones that are being synthesized), including the MMG and TMG series, combined with mutational analysis, to study enzyme kinetics (by means of HLPC and fluorescence methods) to elucidate the dinucleotides' structure-function relationships.
Last updated August 2008
