Melissa Moore - US grants

The removal of introns by RNA splicing is an essential step in the expression of almost all human genes. Many nascent transcripts are subject to alternative splicing, which provides a means for making more than one protein from a single gene. Such alternative splicing can be either developmentally or tissue-specifically controlled. Whether or not an RNA is spliced is also subject to regulation: controlling the level of spliced and unspliced viral RNA in the cytoplasm is critical to the replication of HIV. Thus a detailed knowledge of the splicing process will be essential for better understanding of the basic mechanisms of gene expression, as well as organismal development, oncogenesis and the progression of retroviral infection. Because spliceosomal processing and the self-catalyzed excision of group II introns occur via the same two-step pathway, the two intron types are thought to be evolutionarily related. Therefore, the spliceosome has long been suspected to be an RNA catalyst. However, the precise makeup of the catalytic center(s) has remained elusive, primarily because spliceosomal components that interact directly with the splice site phosphates have yet to be identified. Neither is it known to what extent the active sites for the two steps of splicing overlap, although growing evidence supports some sort of structural rearrangement between the two steps. Even less is known about the catalytic mechanisms and active site structure of group II introns. Thus, whether the common two-step pathway has resulted from divergent or convergent evolution of these intron types remains debatable. Additionally, what mechanistic similarities exist, if any, between the above introns and introns of the group l self-splicing class are totally unknown. A long-term goal of this laboratory is to elucidate the catalytic mechanisms and active site machinery utilized by the spliceosome and group II introns to catalyze intron excision. This proposal focuses primarily on the exon ligation step. Parallel approaches to be used in both systems include: (l) development of a comprehensive kinetic framework for exon ligation using an assay in which the 3' splice site is added in trans; (2) use of chemically modified 3' splice site RNAs to determine the exact substrate structural requirements for exon ligation, and how these structures contribute to splice site recognition and catalysis; and (3) incorporation of photo- and affinity-crosslinking reagents to identify active site components closely juxtaposed to the phosphodiester backbone at the 3' splice site. Together, these experiments should allow direct comparison of active site structure, 3' splice site recognition and mechanisms of catalysis by the spliceosome and both group II and group I self-splicing introns.

Faculty in the Departments of Biochemistry, Chemistry and Biology at Brandeis University are requesting funding for a MALDI-TOF (Matrix- Assisted Laser Desorption Ionization Time-Of-Flight) mass spectrometer. This instrument will be placed in the Core Biomolecules Synthesis and Analysis Facility. It will be freely accessible to all NIH-supported researchers in the School of Science at Brandeis, as well as others on a time-available basis. Currently there is no mass spectrometer at all at Brandeis; at the present time the 13 investigators in the major user group must seek off-campus collaborations to carry out any mass spec analysis of any variety. Therefore acquisition of a state-of-the-art MALDI-TOF mass spectrometer will significantly expand the life science research capabilities at this institution. Immediate uses proposed by the major user group include routine analysis of the products from organic and bioorganic synthesis (e.g. synthesis of natural products, modified nucleic acids, functionalized proteins, enzyme inhibitors and ion channel blockers), mapping sites of post-translational modifications in proteins, monitoring changes in phosphorylation states of proteins, rapid sequencing of new proteins and peptides, monitoring isotope labeling of proteins for NMR analysis, monitoring metal ion replacement in metalloproteins, mapping sites of protein modification by active site-directed inactivators of enzymes and kinetic analysis of peptide bond cleavage by proteases. Institutional commitment for acquisition and maintenance of a state-of- the-art MALDI-TOF is very strong. In particular, the University will provide for the salary of the Principle Operator, as well as guarantee funds for maintenance of the instrument into the foreseeable future.

9977556

Abstract

This project involves the acquisition and installation of a 300 kEV field emission gun (FEG) transmission electron microscope (TEM) to determine the structures of cellular machinery. Cellular machines such as the actin cytoskeleton are often large structures difficult to study by methods other than electron cryomicroscopy. For example, it has been possible to dock atomic models for the cytoskeletal components obtained by x-ray crystallography into the molecular maps of the cytoskeletal complexes obtained by electron cryomicroscope. Analysis of filamentous structures has been extended to 10 A resolution in many cases. A state of the art microscope is essential for these studies. The microscope to be obtained and installed during this project will be utilized for the study of the splicesome, voltage-gated ion channels, the actin bundle of the intestinal microvillus, the bacterial flagellar motor and filament, the bacterial gas vesicle, the receptor-kinase signaling complex and the complex of actin with myosin. The aim of these studies are atomic models of the structures. In some instances the structure will be obtained to atomic resolution using electron cryomicroscopy alone. In other cases, atomic models obtained by x-ray crystallography will be docked into molecular maps obtained by cryomicroscopy. The 300 kEV FEG TEM is essential to these projects because the quality of the micrographs limits the resolution of the map in the same way that the quality of the objective lens limits the resolution of the light microscope. No matter how sophisticated, computer processing cannot extract what is not present in the images. The two key features of this microscope in this regard are the higher voltage and the FEG. With these, it is possible to take highly defocused images which have strong contrast at low resolution but which preserve high resolution detail. The strong contrast at low resolution is essential for accurate alignment of images prior to averaging. If the alignment (or correction for distortion) is not accurate, one will average non-equivalent features and resolution will be lost. This microscope will extend the resolution of current maps and therefore, the ability to obtain accurate atomic models whether it be directly or by the docking of atomic models into molecular maps.

The cryomicroscope is essential to the training of young scientists. Since the kind of microscopy being used represents a frontier in structural studies of cellular machinery, students and post doctoral fellows must be trained in these techniques. The need for such scientists is increasing rapidly now that the power of cryoelectron microscopy has been demonstrated. There are currently few places equipped for this kind of training.

introns; spliceosomes; ribozymes; active sites; RNA splicing; enzyme mechanism; precursor mRNA; thiophosphate; pyrimidine nucleotides; enzyme substrate; chemical cleavage; chemical kinetics; thermodynamics; esterification; polynucleotides; nucleic acid sequence; nucleic acid structure; small nuclear RNA; RNA; divalent cations; affinity labeling; nucleic acid chemical synthesis; crosslink;

This IGERT award establishes a multidisciplinary graduate training program of education and research in the Physics and Chemistry of Biological Systems at Brandeis University. The program will ensure that a) biologists can work effectively with rigorous quantitative methods, new technologies and models, b) physicists and chemists obtain hands-on experience with biological systems and methods, and c) students with a variety of backgrounds learn multiple scientific languages so that they can communicate and work with investigators with skill sets and training different from their own. Graduate students will be carrying out state-of-the-art research in a wide variety of topics including protein complexes, signal transduction and transcription, neuronal networks, biological oscillators, and cognitive processes and behavior. Trainees thesis research will involve quantitative approaches to a biological problem. The educational plan includes laboratory rotations and courses that include modeling and quantitative methods; several new courses will be developed specifically for this program. Trainees will also participate in a semester-long course on the responsible conduct of research, invite and host outside seminar speakers, participate in journal clubs and serve as teaching assistants. To enhance the broader impacts of the grant, trainees will receive formal training in presenting science to lay audiences at two area science museums, and/or through several campus-based educational outreach programs. The IGERT program will provide a free Saturday morning lecture series for local high school teachers, students, and the interested public. Undergraduate minority students will be acquainted with research opportunities at Brandeis. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing, an essential step in eukaryotic gene expression. Human genes are on average >90% intron, and a significant percentage of genetic diseases arise from mutations that that alter splice site choice. It has been estimated that ca. half of of human genes are additionally subject to alternative splicing, which significantly increases the complexity of the proteome encoded by our surprisingly small genome. This alternative splicing is often tissue-specifically or developmentally regulated. The extent to which a transcript is spliced is also subject to control - for example, altering the ratio of spliced and unspliced viral RNA is critical to the replication of HIV. Thus a detailed working knowledge of the splicing process will be essential if we are to understand not only the basic mechanisms of eukaryotic gene expression, but also how they relate to the complex processes of development, oncogenesis, human genetic disorders and the progression of retroviral infection. Studies in this proposal will address three important questions: (A) What is the detailed three-dimensional architecture of the human spliceosome, the macromolecular protein: RNA machine that mediates intron excision?; (B) What is the detailed three-dimensional architecture of the exon junction complex (EJC), a key regulator of spliced mRNA metabolism?; and (C) By what mechanism does the EJC remain stably bound to spliced mRNA? Techniques utilized will include (i) structure determination of purified complexes by cryo- electron microscopy (EM); (ii) labeling of those structures with EM-visible probes to map the locations individual components; (iii) a new methodology for determining relative protein stoichiometries by quantitative mass spectrometry; (iv) protein-protein interaction studies; and (v) determination of the kinetic and thermodynamic properties with respect to RNA and ATP binding by the protein thought to serve as the EJC anchor in the presence or absence of its binding partners. [unreadable] [unreadable] [unreadable]