Marlene Belfort - US grants

Mutational analysis of thymidylate synthase (TS) is proposed to probe structure-function relationships of this metabolically important enzyme. The synthase provides the cell with dTMP, a vital precursor specific to DNA synthesis. Because of its pivotal role in DNA replication TS is a target enzyme in chemotherapy. In order to understand these processes in greater depth it is of interest to study the regulation and mechanism of action of this enzyme. The overall objective of this study is to provide a genetic complement to the biochemical and structural studies being conducted on the synthase. As a basis to the proposed mutational analysis and having chosen the E. coli enzyme as a model system, we established the nucleotide sequence of the thyA gene, and the amino acid sequence of its TS product. Positive selection for thy- cells has allowed us to isolate a wide spectrum of random mutations after in vitro mutagenesis of the cloned gene. Rapid biochemical screening procedures as well as genetic and kinetic analyses of these mutants will more sharply define functional regions and will guide site-specific mutagenesis strategies to probe the precise involvement of particular residues in the catalytic process. The ability to construct strains which overproduce mutant enzyme will facilitate biochemical and structural studies in this and other laboratories. This combination of the genetic and biochemical approaches will help relate mutational change at the nucleotide and amino acid levels to altered kinetic and physical properties of TS. The ultimate goal is the delineation of those features and domains of TS which underly catalysis and are variously involved in substrate and cofactor binding, subunit folding, dimerization and multienzyme complex formation.

The long-term objective of this research is to use the split td gene of phage T4 as the molecular paradigm for understanding the RNA and DNA transactions of a self-splicing, mobile intron. During the first 5-year funding period (supported by NSF grant DMB8502961) we established that the td intervening sequence is a group I self-splicing intron. We used this knowledge to detect two other introns in T4 and discovered that one of these as well as the td intron are mobile, transferring efficiently to intronless alleles of their respective genes. As for their eukaryotic group I counterparts, intron mobility is dependent upon an endonuclease encoded by the intron itself. The proposed research is intended to further our fundamental understanding of aspects of both the RNA-based (splicing) and DNA-based (mobility) processes, using the td gene as model system. To both ends we shall continue to exploit the powerful positive and negative selections provided by this phage genetic system and by the td gene in particular. For the splicing studies we shall continue to use molecular genetics to analyze the function of a prokaryotic ribozyme. We shall conduct parallel studies to investigate the possible role of Escherichia coli functions as accessories to the self-splicing process. With respect to DNA mobility, we shall examine the function of the td endonuclease, which mediates the process, and its interaction with its DNA target, the intron homing site. We shall also ask some more general questions about the intron-phage relationship and use the td gene as a model for exploring the possibility of intron loss in a prokaryotic system. Finally, to rationalize the existence of introns , in streamlined phage genomes, we wish to address the hypothesis that introns provide a selective advantage by enhancing phage recombinogenicity. Together, the proposed experiments represent a continuation of ongoing work that exploits facile prokaryotic genetic techniques to shed light on the multifaceted reactions of group I introns, which are dynamic genetic elements in common to the pro- and eukaryotic kingdoms.

This conference will bring together an international group experts on various aspects of RNA function. Some of these aspects are newly emerging areas of intensive research, and one of them has never before been the subject of a formally convened session. The series of conferences to which this one belongs are noted for their promotion of lively exchange of ideas, as well as the unusual opportunity they have provided for younger scientists (students and postdoctoral fellows) to participate in face-to-face discussions with acknowledged leaders in the field. The discussions at this conference will permit exchange of ideas among experts who would not normally interact, and should thus provide a great impetus for research in this exciting field.

9318966 Wickens This award will support an annual meeting of researchers studying the processing of RNA molecules. The post transcriptional modification of RNAs by a variety of mechanisms including splicing, polyadenylation, base modification, nuclease trimming, and editing is crucial to their biological activities. The meeting has been held annually for over 10 years and has played a key role in stimulating research in this broad, important field. %%% The biosynthesis of RNA from DNA is the first step in utilizing genetic information. The RNA is almost always modified in one or more ways before the cell can actually use these molecules for the many functions which they serve. This award supports a conference of researchers who study the modifications of RNA molecules which transform them into key components of the cellular machinery. ***

9322134 Mannella This award provides funds for renewal of a summer REU program held at the Wadsworth Center for Laboratories and Research (WCL&R) in Albany, NY. The research scientists who supervise the students in this program are also faculty in the Department of Biomedical Sciences of the State University of New York at Albany's School of Public Health. The WCL&R is a unique laboratory with state-of-the-art facilities, in which basic research programs have developed alongside mission-oriented public health laboratories. The research interests of the participating BMS faculty cover a broad range, with emphasis in molecular genetics, cell biology and structural biology. Eleven undergraduate students interested in research careers are recruited from colleges in the Northeast and from historically Black colleges in the South. Another 5 students participate in the program with stipend support from the WCL&R. Laboratory projects in basic biological research topics are chosen that are interesting, likely to yield results within the time-frame of the program, and allow for some degree of student development and independence. Weekly group meetings are used to discuss science- and career-related issues and to monitor student progress. The students make short oral presentations of their work in a symposium in the last week of the program, and also submit written reports before they leave, to stress the importance of communication skills within science. ***

9802699 Belfort This meeting will cover the structure, mechanism, and biological roles of enzymes that act on DNA and RNA. Included are both protein and RNA enzymes that act on nucleic acids. The meeting is designed to attract a broad cross-disciplinary spectrum of scientists from the U.S. and abroad. These include chemists, biochemists, structural and molecular biologists, geneticists and cell biologists. This meeting will be the third held by the Federation of American Societies of Experimental Biology (FASEB) on this general topic, the last having been in 1996. Since then, the understanding of the structure, mechanism and regulatory potential of these molecules has expanded greatly . This FASEB meeting is therefore both timely and topical. The organizers have selected discussion leaders for the nine sessions and a keynote speaker for an additional session. Speakers range from Nobel prize-winners at the forefront of their fields to accomplished young scientists destined to make inroads in their specialities.The topics to be covered at the meeting include nuclease structure & function; synthetic enzymes and novel activities; endonucleases and integrases of mobile elements; catalysis of and by RNA; recombination and repair; ligases, topoisomerases, and capping enzymes; replication proteins; and genomics. In addition, there will be a poster competition; students and post-docs with winning posters will be given the opportunity to present a talk. The meeting will provide a focus for powerful and fruitful approaches to studying fundamental features of nucleic acids and will serve as a multidisciplinary forum for the study of enzymes which interact with nucleic acids. Although meetings centering on various specialities of this general field are also held, this particular interdisciplinary mix of topics is unprecedented and promises a unique opportunity for fertile scientific exchange among scientists with interests in basic research and biotechnology.

? DESCRIPTION (provided by applicant): Catalytic RNAs and retrotransposons are key RNA elements that help shape genomes. Bacterial group II introns (gII-ins) are both self-splicing RNAs, which are the putative progenitors of spliceosomal introns, and mobile retroelements, and as such they occupy a pivotal role in early eukaryotic evolution. At a structural level, gII-in RNA combines with an intron-encoded protein to form a ribonucleoprotein (RNP) that is active in both splicing and mobility. The overall goal of this application is to understand the structure and function of these bacterial mobile self-splicing retroelements, while relating them to their eukaryotic spliceosomal and retroelement counterparts. This goal will be achieved by combining interdisciplinary biochemical, biophysical, cellular, computational, genetic, structural and systems approaches. Considerable progress over the past funding period, including structural analysis of the gII-in RNP, and functional studies relating retrotransposition to conjugation, is te springboard for the proposed specific aims: Aim 1: To capture structure transitions of the gII-in RNP in splicing and gene targeting. We will use native RNPs purified from L. lactis, for which we have derived a 4.5 Å cryo-EM structure, to develop a series of structural and kinetic snapshots of the gII-in RNP at different stages. These include the loose RNP precursor; the compact, spliced free intron RNP; and the free intron RNP attacking its DNA target for retromobility. Emphasis will also be placed on modification of the intron RNA and its role in catalysis of splicing and retromobility, structure transitions and interactions with the intron-encoded protein. Finally, our recent discovery that the modular intron-encoded protein is similar to two pivotal eukaryotic proteins, Prp8, the most conserved protein in the spliceosome, and telomerase reverse transcriptase, which preserves chromosome ends, is the basis of tests of analogous functions in splicing and retromobility. Aim 2: To understand host-retrotransposon relationships across kingdoms. We will further investigate the common residence of gII-ins with other mobile elements, to determine their interrelationships with the bacterial mobilome. We will also define the dynamic relationship, both positive and negative, between the parasitic gII-in and the bacterial host, L. lactis, using biochemical, genetic and systems approaches. These studies will be integrated with the NIH-funded Center for Systems Biology of Retrotransposition, which focuses on mammalian retrotransposons. The impact of our interdisciplinary approach is an enhanced understanding of the structure and function of self-splicing elements that have been exploited biotechnologically to edit genes, and which resemble retrotransposons that sculpt diverse genomes in health and disease.

DESCRIPTION (provided by applicant): Self-splicing introns and inteins attract attention for their molecular mechanisms, phylogentic diversity, role in genome evolution, and application in research, biotechnology and medicine. Interest in these elements stems from their self-splicing properties at the RNA level for introns and protein level for inteins, and from their ability to ac as mobile genetic elements at the DNA level. In the past funding period, we made considerable progress in structural and functional characterization of these self-splicing introns and inteins. For the next research phase, we will focus on the relatively understudied inteins. Inteins exist at the crossroads of the disparate disciplines of protein chemistry, biotechnology and molecular evolution. Their autocatalytic peptide cleavage and ligation reactions make them useful tools in modern chemical biology, whereas their existence within proteins critical to vital cellular processes raises provocative questions about their function in nature. We propose the following three specific aims, based on discoveries made in the past funding period: In the first aim, we will analyze the role of the flanking host sequences, the exteins, on intein structure, splicing an evolution. This work is enabled by our collaborations with physicists and structural biologists. We will also address a bold hypothesis, that inteins persist in specific exteins because they confer a selective advantage on their host, through adaptive interactions with flanking extein residues. In the second aim, we will study intein inhibitors as mechanistic probes and antimicrobials. Thus we will exploit the existence of inteins in critical genes of microbial pathogens, to probe inteins as novel targets for bacterial and fungal antibiotics. We will further characterize cisplatin, the chemotherapeutic agent, which we identified as a protein splicing inhibitor. We will investigate cisplatin's efficacy against infection by Mycobacterium tuberculosis and also test its ability to curtail activity of cryptococcal inteins. Additionally, we aim to isolte small-molecule and peptide inhibitors, with a view to comparing their properties with each other and with cisplatin. In the third aim, we will use molecular methodologies previously developed in our lab (redox traps, gain-of-fluorescence protease sensors, and phage display selections), to fashion tools for biotechnology and medicine. Thus, we will exploit our ability to isolate wild-typ intein precursors for biological and chemical applications, and construct sensors for proteases in a botulism toxin diagnostic and to detect tuberculosis (TB) biomarkers as a TB diagnostic. Once again we are taking collaborative, interdisciplinary approaches, which combine genetics, biochemistry and microbiology with physics and structural biology. In this way, we will enhance our understanding of the structure, function and evolution of inteins, as a means to exploit them as potential targets for drug development and as novel reagents in biotechnology and medical diagnostics. PUBLIC HEALTH RELEVANCE: The overall goal of this application is to build upon progress made in the previous funding period, using interdisciplinary approaches to study intein structure, function, evolution and application. The applied aspects of the proposal relate to isolation and characterization of inhibitors of microbial inteins, as a means to discover novel antibiotics against tuberculosis and mycoses. We will also exploit intein technology to develop a diagnostic sensor for botulinum toxin and for tuberculosis biomarkers.

? DESCRIPTION (provided by applicant): Catalytic RNAs and retrotransposons are key RNA elements that help shape genomes. Bacterial group II introns (gII-ins) are both self-splicing RNAs, which are the putative progenitors of spliceosomal introns, and mobile retroelements, and as such they occupy a pivotal role in early eukaryotic evolution. Yet, gII-ins are mysteriously absent from nuclear genomes, where we have shown them to silence gene expression. At a structural level, gII-in RNA combines with an intron-encoded protein to form a ribonucleoprotein (RNP) that is active in both splicing and mobility. The overall goal of this application is to understand the structure and function of these bacterial mobile self-splicing retroelements, while relating them to their eukaryotic spliceosomal and retroelement counterparts. This goal will be achieved by combining interdisciplinary biochemical, biophysical, cellular, computational, genetic, structural and systems approaches. The work will be performed in an array of model organisms, including the bacterium Lactococcus lactis and the yeast Saccharomyces cerevisiae. Considerable progress over the past funding period, including structural analysis of the gII-in RNP, and functional studies in bacteria and yeast, is the springboard for three proposed specific aims: Aim 1: To capture structure transitions of the gII-in RNP in splicing and gene targeting. We will use native RNPs purified from L. lactis to develop a series of structural and kinetic snapshots of the gII-in RNP at different stages. These include the loose RNP precursor; the compact, spliced free intron RNP; and the free intron RNP attacking its DNA target for retromobility. Aim 2: To define silencing of gII-in-containing genes in eukaryotes. We will dissect the process of silencing of gene expression imposed by the gII-in, which is thought to have played a role in the gII- in/spliceosomal intron transition, by defining host factors that result in RNA mislocalization. Thus, by combining yeast genetics, RNA biochemistry, cell and systems biology, we will establish a mechanistic appreciation of why gII-ins were expunged from eukaryotic nuclei. Aim 3: To understand host-retrotransposon relationships across kingdoms. We will define the dynamic relationship, both positive and negative, between the parasitic gII-in and the bacterial host, L. lactis, using biochemical, genetic and systems approaches. These studies will be integrated with the NIH- funded Center for Systems Biology of Retrotransposition, which focuses on mammalian retrotransposons. The impact of our trans-kingdom approach is an enhanced understanding of the structure and function of self-splicing elements that have been exploited biotechnologically to edit genes, and which resemble retrotransposons that sculpt diverse genomes in health and disease.

1. PROJECT SUMMARY The overall goal of the RNA Fellows program is to rigorously train graduate students for careers in science and technology companies, science communication organizations, public service or academia. Our RNA Fellows will plan and execute RNA centric research projects and actively participate in year-long business plan competitions or writing events, so that they can be qualified as science entrepreneurs or science communicators. These unique experiences will help our students bring their science based training to the pharmaceutical, biotechnology, business start-up, finance, public policy, journalism and other non-traditional fields. Our distinctive T32 training grant is designed to train students for careers outside academia, but it will allow them to excel in traditional post-doctoral environments if they so choose. We will prepare students to harness the potential of RNA science and technology by cross-training in chemical, molecular, nanoscale science, and public health disciplines, as we exploit the intellectual capital and the resources of the RNA Institute with members from University at Albany, SUNY (State University of New York) and SUNY Polytechnic Institute. The RNA Fellows program will give students in four departmental degree programs, Biological Sciences, Biomedical Sciences, Chemistry and Nanobiosciences, unique research and educational experiences that will provide the professional and personal skills to meet the challenges needed for a cutting edge science and technology workforce. Doctoral research projects will extend from the fundamental biology of RNA in gene-expression, health and disease to technology innovation at the interfaces of biology, chemistry, biophysics, computation, nanoscale science, nanoscale engineering and informatics. There are six RNA research themes representing mentor faculty interests (i) Computational Analysis of RNA Structure; (ii) Experimental Analysis of RNA Sequence and Structure; (iii) Technology Development; and three biomedical themes of (i) Basic Cellular Processes; (ii) Infectious Disease; and (iii) Development and Cancer. A two- semester flagship RNA course based on these themes and a course in mathematical or biomedical fluency will be combined with options to train in entrepreneurship or writing, via the Blackstone Entrepreneurship Launchpad at our School of Business or at the renowned NY State Writers Institute, respectively. As evidence of Institutional support, we have implemented a SUNY sponsored RNA Fellows program that has 18 total students from three yearly cohorts. The 33 training faculty have on average over $372K a year in direct grant support and focus on our RNA core themes. UAlbany has a strong commitment to student diversity with 29.9% of its students being under-represented minorities. Together with an existing aggressive recruitment plan, our current demographics and our participation in the New York Louis Stokes Alliance for Minority Participation (LSAMP) positons us to grow our recruitment of individuals from underrepresented backgrounds. Our T32 training grant application requests funding for 20 student slots, which is matched with over $1 million from SUNY in institutional support. !

The University at Albany is a doctoral institution with a strong commitment to research excellence. It boasts a highly diverse undergraduate population and strives to excel as a diverse and inclusive campus community; yet, women and women of color are underrepresented in the faculty ranks of STEM departments. The goal of Project SAGES is to create an environment in which women of all backgrounds and identities can thrive and develop their careers to their fullest potential. To accomplish this goal, Project SAGES seeks to increase the number of women scientists in STEM fields through proactive recruitment and unbiased hiring procedures and retain them by creating a climate and culture in which women feel supported, thrive, and advance in their careers from assistant to associate to full professor.

The aims of Project SAGES are threefold: Aim 1 seeks to increase the diversity of applicant pools for faculty searches in STEM through a postdoctoral visitation program to identify competitive candidates for job vacancies and by education of search committees and decision makers on inclusive, unbiased search processes. To promote diverse applicant pools, search chairs will receive real-time feedback about pools to permit proactive measures if diversity does not meet national norms for the field. Aim 2 seeks to improve the campus climate by subtly shifting departmental norms and cultures. Departmental interventions will comprise awareness training for chairs, the formation of department climate committees, and ally training for men and women, all with the goal of creating a more inclusive environment. In addition, consistent and transparent policies and procedures will be implemented that meet the needs of a diverse faculty. Aim 3 seeks to support women’s research success. A networking program for women faculty and women of color and a pilot funding program will allow women in STEM to build collaborative research teams. Additional support is provided with a novel external sponsor program in which female assistant professors are paired with a prominent expert from another university who will provide ongoing guidance on best strategies for networking, funding, publishing, and achieving tenure. Through these interventions, Project SAGES aims to bring about sustained institutional transformation at the University at Albany with the goal of achieving inclusive excellence in STEM that better reflects the university’s highly diverse student body. The interventions will lead to a more nurturing and family-friendly environment for women scientists of diverse identities and maximize their success through comprehensive professional support structures and an equitable tenure and promotion process. Outcomes from Project SAGES will be disseminated through multiple outlets including a website. The NSF ADVANCE program is designed to foster gender equity through a focus on the identification and elimination of organizational barriers that impede the full participation and advancement of diverse faculty in academic institutions. Organizational barriers that inhibit equity may exist in policies, processes, practices, and the organizational culture and climate. ADVANCE "Adaptation" awards provide support for the adaptation and adoption of evidence-based strategies to academic, non-profit institutions of higher education as well as non-academic, non-profit organizations.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.