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Feigon Lab RESEARCH PROJECTS |
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Nucleic acid structure and function RNA has increasingly been found to have a role in everything from regulation of transcription and translation to synthesis of the ends of chromosomes to enzymatic reactions. In order to do these things, RNA assumes a variety of shapes and often forms specific RNA-protein complexes. DNA can also adopt a variety of conformations besides the Watson-Crick double helix, and its interactions with proteins are vital to regulation of replication, transcription, and repair. In the Feigon laboratory, the structure and function of DNA and RNA are being studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy, which provides a method for determining the three-dimensional structures of macromolecules and to study their dynamics in solution. Larger complexes are also being studied by X-ray crystallography. Structural information is correlated with function using a variety of biochemical and molecular biological techniques, to provide insight into how these nucleic acids and nucleic acid-protein complexes work in the cell and how mutations in some RNAs and DNAs can lead to disease. Eukaryotic ribosome biogenesis—nucleolin, RNase III, and H/ACA snoRNPs One current focus of the research in the group has to do with events taking place in the nucleolus. The nucleolus is a dynamic structure that assembles around the transcribing ribosomal DNA. While the nucleolus is known primarily as the site of ribosome transcription and assembly, it has been more recently implicated to play important roles in cellular aging and cell cycle control. Nucleolin is the most abundant nucleolar protein and is involved in many steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, ribosome assembly and nucleocytoplasmic transport. The specific and transient interaction of this multidomain protein with nascent pre-rRNA and ribosomal proteins is thought to be important for the proper folding of pre-rRNA and its packaging into pre-ribosomal particles. Recently, nucleolin has been implicated to have a role in mRNA stability and to interact with telomerase. We recently completed the structure determination of nucleolin RNA binding domains 1+2 (RBD12) and with its natural RNA target sequences. Our studies of the interaction of nucleolin with both in vitro selected and natural substrates has provide important insights into how multiple RBD proteins use both their RBDs and linker to recognize specific sequences.
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RNAses III are an important class of RNA cleaving enzymes that are involved in RNA processing and regulation of gene expression. The yeast (S. cerevisiae) RNAse III, Rnt1p, recognizes snRNA, snoRNA, and rRNA substrates that contain RNA hairpins with a 4 nt loop in which the first nt is usually an A and the second nt is always a G, and cleavage occurs 14-16 bp from the loop. We have been conducting structural and functional studies to determine how Rnt1p recognizes its substrates. Our recent work on a complex of Rnt1p dsRBD with its substrate showed that binding involves recognition of a pre-formed loop conformation and was the first dsRBD complex with a natural substrate. This work has important implications for how other RNAse IIIs may target siRNA and miRNAs. |
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Another major part of this project is to study the structures of box H/ACA snoRNPs. These contain the 'guide' RNAs that are involved in pseudouridylation of sites on the rRNA.
Structure and function of telomeres and telomerase We are interested in the structure of telomeres and their synthesis by telomerase. One of the oldest projects in the lab is the study of quadruplex structures formed by telomere repeat oligonucleotides. Telomeres are the physical ends of chromosomes, and are composed of a 1000s of repeats of a short G-rich (in one strand) sequence of DNA and associated proteins. Telomeres generally contain a single strand overhang of the G-rich repeat. We determined the first DNA quadruplex structure in 1992, formed by the telomere repeat oligonucleotide d(G4T4G4) from Oxytricha nova, and showed that it is a dimeric symmetric quadruplex with 4 G quartets and thymine loops which span the diagonal of the end quartets. We have extensively studied structures and cation interactions with various quadruplexes, and are currently studying the effect of cations on the hydrogen bond lengths.
Recently, we have turned our focus to telomerase structure and function. The telomere repeat sequence is not replicated by the normal DNA replication machinery, but rather by a unique enzyme complex called telomerase. Telomerase uses an RNA template that is part of the enzyme and a specialized reverse transcriptase to processively synthesize the G-rich strand. Telomerase has only a low or undetectable level of activity in normal somatic cells, but is up-regulated in most cancers, and is thus of interest as a target for anticancer drugs. In addition, mutations in the telomerase RNA have been found to cause some forms of the genetic diseases dyskeratosis congenita and aplastic anemia. Human telomerase contains a 451 nt RNA along with a variety of proteins besides the telomerase reverse transcriptase. Only about 10 nt are needed for the template, so what is the function of all that extra RNA? This is the question that we trying to answer, by investigating the structure of various domains of the telomerase RNA. We recently reported the first structure of a sub-domain of telomerase RNA, a portion of the so-called pseudoknot domain. We found that the pseudoknot is in equilibrium with a hairpin, which adopts a unique structure containing a short pyrimidine paired helix. Based on the structure, thermodynamics, phylogenetic analysis, and biochemical data, a conformational switch in the pseudoknot domain was proposed to be important for the enzyme activity. A mutation in the RNA linked to dyskeratosis congenital shifts the pseudoknot-hairpin equilibrium to the hairpin form. |
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Interactions of HHR23A with cellular DNA repair proteins, the ubiquitin/proteosome pathway, and HIV-1 Vpr HHR23A, the human homologue of Rad23, is a multidomain protein containing an N-terminal Ubl (ubiquitin-like) domain, two UBA (ubiquitin associated) domains, and an XPC (Xeroderma pigmentosum complex C) protein-binding domain. Although HHR23A was first identified as a component of the DNA repair XPC complex, involved in nucleotide excision repair, recent studies also link it to the ubiquitin/proteosome pathway. The HIV-1 gene, vpr, encodes a 96 amino acid protein that is required for efficient infection of nondividing cells such as macrophages. Vpr induces cell cycle arrest at the G2/M checkpoint leading to subsequent apoptosis. Vpr interacts with a number of cellular proteins, including HHR23A. Work by our collaborators in the I.S.Y. Chen lab at UCLA has provided support for the hypothesis that the Vpr/HHR23A/B interaction may be a key step in Vpr mediated cell cycle arrest. We determined the structure of the C-terminal UBA domain that interacts specifically with Vpr, and showed that mutation of a critical proline residue completely abolishes this interaction. Our current work on this project is focused on structure determination of other domains of HHR23A and their complexes with interacting proteins involved in DNA repair and ubiquitin/proteosome pathway. We have determined the structures of both of the UBA domains, the XPC-binding domain, and the Ubl domain. We have shown that the UBA domains interact with the b-sheet of ubiquitin via a conserved hydrophobic surface. We also recently reported the structure of a complex the Ubl domain with a UIM domain of the S5a subunit of the proteosome. This was the first structure of a UIM bound to a member of the ubiquitin family, and revealed the molecular determinants of the Ubl-proteasome interaction. The overall objective of these studies is to develop a comprehensive understanding of the structure/function relationships of HHR23A/B in cellular function, particularly as it relates to DNA repair, cell cycle, and ubiquitin/proteasome pathways. This understanding will, in turn, provide us with critical information needed to understand the role of the Vpr/HHR23A interaction in apoptosis. |
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Chromatin remodeling and DNA repair Non-histone protein 6A (Nhp6a) is an abundant chromatin-associated protein from Saccharomyces cerevisiae that belongs to the HMGB family of non-specific DNA binding proteins. This class of HMG proteins, typified by vertebrate HMG1 and HMG2 that contain two HMG boxes, are present at a level of one molecule per 2-3 nucleosomes and thus are a major constituent of eukaryotic chromatin. Although their biological functions are just beginning to be revealed, they have been shown to participate in reactions as diverse as DNA recombination, repair, activation and repression of transcription as well as nucleosome assembly and disassembly. The HMGB proteins strongly distort DNA upon binding and can stabilize bent and supercoiled DNA. This DNA architectural role in facilitating the formation of higher order nucleoprotein complexes is believed to be a critical component of their activity in many of these reactions. We recently completed the refined structure of a complex of Nhp6a and DNA. A major goal was to understand how these 'sequence neutral' proteins target specific DNA sites. Our structural and mutational analysis as well as comparison of the DNA in the complex to the free DNA provided insight into the factors that contribute to binding site selection and DNA deformations in the complex. The HMG proteins also bind to DNA that is distorted by agents such as the anti-cancer drug CisPt. The specific interaction of HMG proteins with cisplatin DNA is thought to affect the DNA repair machinery of the cell and therefore plays a role in the mode of action of these drugs. We have used a combination of mutagenesis, gel mobility shift assays, footprinting, and NMR to probe the interaction of Nhp6a and cisplatin-modified DNA in vitro, and the relationship of Nhp6a/b to cisplatin cytotoxicity in vivo. |
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Cation binding to nucleic acids |
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Cations play essential roles in nucleic acid structure, stability, folding, and catalysis. Nevertheless, the interactions between mono- and multivalent cations and nucleic acids are in many cases not well understood. We have been using both new (developed in our lab) and established NMR methods to study the interaction of cations with DNA and RNA. We identified specific monovalent cation binding sites on both DNA quadruplexes and on duplex DNA. One major goal is to understand how cations affect the structure of DNA A-tracts. Our laboratory was the first to show experimentally that cations could bind to specific sites within DNA A-tracts. We recently determined the solution structures of two DNA A-tracts with NH3+ |
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counterions, and used these structures to create models of these DNAs in the context of 15 helical repeats. This NMR based structural modeling of d(CAAAATTTTG) and d(GTTTTAAAAC)15 surprisingly revealed that the former forms a left-handed superhelix with a diameter of 110Å and a pitch of ~80Å, similar to the DNA in the nucleosome, while the latter has only a gentle writhe and therefore appears nearly straight. Furthermore, the gel electrophoretic mobility of these DNA oligomers shows a monovalent cation dependence, providing further support that they play a fundamental role in DNA A-tract bending. We are also studying the role of cations in RNA structure and ribozyme catalysis.
Methods development for NMR (and EPR) studies of nucleic acids Many of our studies of nucleic acids have necessitated the development or optimization of NMR techniques for spectral assignment and structure determination of nucleic acids. We have an ongoing collaboration (since 1989) with Professor Vladimir Sklenar (Masaryk University, Brno, Czech Republic) to develop NMR methods for studying nucleic acids. Our current focus is on optimizing methods for studying RNA-protein complexes. |
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We have also collaborated with the laboratory of Stephen Grzesiek to study hydrogen bonding in nucleic acids, particularly DNA triplexes and quadruplexes, using measurements of scalar coupling constants between nuclei involved in hydrogen bonds.
In collaboration with Profs. Peter Qin and Wayne Hubbell, we have been developing and applying EPR site-directed spin labeling techniques to study the structure and dynamics of RNA, specifically the GAAA tetraloop receptor both free and bound to its receptor. This interaction is a frequently occurring motif that is important for long-range tertiary interactions in RNA. These methods will also be applicable to studies of larger RNAs.
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