Venkat Gopalan - US grants

0091081 Venkat Gopalan
The long term goal of this research is to understand RNA-protein interactions in Escherichia coli RNase P, an enzyme essential for the processing precursor tRNAs (ptRNAs) to their mature forms. E. coli RNase P, a ribonucleoprotein (RNP) complex, consists of a catalytic RNA subunit (M1 RNA) and a protein cofactor (C5 protein); both subunits are essential for its catalytic activity in vivo. The role of C5 protein in RNase P catalysis is distinct from other protein-facilitated RNA-catalyzed reactions in that it enhances the catalytic efficiency and versatility of a catalytic RNA which acts in trans on numerous substrates. Recent studies have demonstrated that the protein not only enhances the affinity of the substrate for the catalytic RNA subunit but also increases the rate of the RNA-catalyzed cleavage reaction. Several questions with regard to the mechanism of action of this unique catalytic RNP complex remain unanswered. In this project, a combination of biochemical and biophysical approaches will be used to determine the role of C5 protein in assembling a functional RNase P complex. The first objective of this study will be to employ structure-based mutagenesis and a genetic complementation assay to identify amino acid residues in C5 protein that are essential for its function in vivo. The second objective is to dissect the mechanisms by which the protein cofactor can exert effects on substrate recognition as well as catalysis. Lastly, by rational design of C5 mutants bearing cysteine residues at various positions in the protein molecule and modifying the cysteine residues with thiol-specific crosslinking, footprinting and spectroscopic probes, low resolution information regarding contact sites between (i) C5 protein and M1 RNA, and (ii) C5 protein and ptRNA substrates, will be obtained and used to gain structural perspectives critical for elucidating the mechanism of action of RNase P. RNA-protein interactions play an important role in numerous regulatory systems in vivo (e.g., translational control of gene expression). Results from this study will serve as a paradigm for understanding intermolecular interactions in other RNPs that control various prokaryotic and eukaryotic cellular processes. Most of the experiments described above are in progress and will rapidly furnish valuable insights. In addition to integrating laboratory research and education for several undergraduate and graduate students, this project has already helped renew and nurture several academic collaborations.

Ribonuclease P (RNase P) is a ubiquitous and essential ribonucleoprotein (RNP) involved in tRNA biogenesis. Bacterial RNase P consists of a catalytic RNA subunit and a protein cofactor. While the RNA subunits of most archaeal and all eukaryal RNase P contain several of the critical and conserved secondary structural elements present in their bacterial counterpart, they are not catalytically active in vitro in the absence of their multiple protein subunits, whose number varies from four to ten depending on the source. Since both the RNA and protein subunits are essential for function in vivo in all organisms, RNase P serves as a paradigm for understanding how proteins modulate RNA function. Moreover, the variations in the functional assignment and composition of subunits in the three domains of life provide a unique opportunity to evaluate if RNase P represents an early RNA enzyme progressing through evolutionary stages to an RNA-protein enzyme in which proteins have usurped most of the functions originally carried out by the RNA. Proteins could have also been recruited to facilitate subcellular targeting and spatio-temporal control of RNase P expression. Any insight in this regard would provide some vindication of the provocative RNA world hypothesis. The long-term goal of this research is to use plant RNase P as a model system for understanding the complex subunit make-up of eukaryal RNase P in a multi-cellular organism and also employ this knowledge to exploit plant RNase P for targeted degradation of endogenous mRNAs, an application that would facilitate discovery of gene-function relationships. This project will help build a network of academic collaborations and strengthen the infrastructure for research at a land grant institution. The dual aims of fostering learning and advancing discovery in biochemistry, plant molecular biology and biotechnology will continue to be accomplished by innovatively integrating state of the art multi-dimensional research training and education for undergraduates, graduates, and postdoctoral scholars.

This REU program at Ohio State University (OSU) will immerse students in an intense yet rewarding 10-week research experience that allows them to discover firsthand the excitement of science. Research projects will involve molecular-genetic and biochemical approaches to problems in biology. After an initial one-week laboratory workshop, which will provide students with a set of basic technical skills, REU participants will spend the next nine weeks working in one of the participating laboratories. Students will investigate a specific research problem and will be encouraged to develop independence in planning and interpreting experiments. At the end of the summer, they will write a summary of their experimental results and present them in a poster format. Students will also attend journal clubs, workshops, and field trips to explore and discuss (i) research, (ii) career options in science, (iii) cutting-edge technology at OSU and in surrounding biotechnology centers, and (iv) ethical issues that they are likely to face as scientists and as citizens. To promote interactions in relaxed settings, there are planned biweekly social events. Students will be recruited from institutions that focus primarily on undergraduate education. Students from groups under-represented in science are strongly encouraged to apply. Indicators of promise, other than GPA, will be taken into serious consideration in selecting participants. More information is available at http://www.biosci.ohio-state.edu/~mgmajors/html/mg_reu.html or by contact Deborah Dotter, (614) 292-8084, at dotter.4@osu.edu.

An emerging challenge pursuant to acquisition of genomic sequences is the need for
efficient tools for specific and regulatable disruption of gene expression to establish gene
function. This SGER project aims to use a strategy for targeted and tightly controlled
degradation of any cellular RNA in plants, using Arabidopsis as a test organism. The method depends on using RNase P, a ubiquitous enzyme involved in tRNA biogenesis, as a reagent that specifically cleaves
an endogenous target RNA and disrupts either its translatability or other biological
function(s). This RNase P-mediated cleavage depends on the expression of another small
RNA called an external guide sequence (EGS), which when bound to the target mRNA
renders the resulting complex a substrate for RNase P. By comparing the efficacy,
sequence specificity, inducibility and cell autonomy of the RNase P- and EGS-based
method versus RNAi in disrupting the expression of target mRNAs of reporters
(luciferase, green fluorescent protein) and tissue-specific transcription factors
(WEREWOLF and GLABROUS1), the pros and cons of these potential gene-function
discovery tools will be elucidated. In addition to serving as a functional genomics tool
for plant biologists, the EGS method could be used to enhance agricultural productivity
by engineering disease resistance or altering metabolic/physiological traits.
Broader impacts of the proposed research .

The proposed studies are based on a solid foundation of promising preliminary results in a monocot and a dicot. The project will enable (i) multi-disciplinary state-of-the-art training for researchers at various stages in their careers, (ii) a network of academic
research collaborations and enhancement of the research infrastructure at a pioneering
land-grant institution, and (iii) the development of a new, exciting technology with
significant payoffs in agricultural biotechnology.

It is anticipated that after two years all the necessary experimental protocols to
help other researchers design their own studies for targeted disruption of gene
expression using the EGS method will become available. To ensure that the results of
this study will have maximum impact in the research community, reagents and tools
needed for the EGS strategy will be provided immediately upon request.

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are genomically-encoded small RNAs (~ 21 nts) that bind to target mRNAs in a sequence-specific manner to regulate their translation and/or decay. In addition to its role in regulating cholesterol and fatty acid metabolism, the abundant liver-specific microRNA miR-122 also plays a central role in the pathogenesis of Hepatitis C virus (HCV) and associated hepatocellular carcinoma by stimulating viral replication through its binding to target sites in the 5'noncoding region of HCV RNA. A number of antisense approaches, which involve the repeated high-dose administration of modified oligonucleotides, have been described for antagonizing miR-122 function. This proposal describes a novel approach using a sequence-specific ribozyme to inactivate miR-122. The advantage of a ribozyme is that it is catalytic;once introduced into the cell, a single molecule will inactivate multiple copies of the miRNA target. Our research design exploits the properties of M1 RNA, the catalytic subunit of Escherichia coli RNase P. The covalent attachment of a guide sequence to M1 RNA generates a customized ribozyme that selectively inactivates the complementary target RNA. We have recently demonstrated the usefulness of this approach to disrupt miRNA function in Arabidopsis plants. Aim 1 will employ both RNA engineering and in vitro evolution strategies to develop an array of M1 RNA-based ribozymes that will effectively and selectively degrade miR-122. Aim 2 will evaluate the efficacy of these ribozymes against miR-122 in Huh-7 liver cancer cells, which have high levels of miR-122. To control the level of ribozyme, it will be expressed from a tetracycline-regulated promoter. Establishing the quantitative relationship between ribozyme expression, the levels of miR-122 and target gene expression will be a cornerstone of this work. RNase protection and qPCR assays will be used to measure the levels of ribozymes and miR-122, respectively. Target gene expression will be monitored using a Renilla luciferase reporter with three miR-122 binding sites from the cationic amino acid transporter 1 (CAT-1) mRNA, qPCR for five endogenous target mRNAs, and by Western blotting for each of these proteins. miRNA microarrays will be used to determine specificity of the ribozyme for targeting miR-122. Lastly, the generality of this approach will be tested by replacing the miR-122 guide sequence in the ribozyme with one complementary to let-7a and thereby targeting let-7a. The overall goal of this R21 proposal is to determine the feasibility of disrupting miR-122 function with a ribozyme, and to lay the groundwork for a subsequent R01 application that will use the tools developed here to directly target HCV. PUBLIC HEALTH RELEVANCE: The replication of Hepatitis C virus (HCV) in liver cells is stimulated by the binding of microRNA-122 (miR-122), the most abundant small noncoding RNA in liver. This proposal describes a new approach to inactivating miR-122 using a catalytic RNA enzyme. The long term goal is to apply this strategy to the treatment of HCV infection.

SUMMARY Our scientific objective is to understand how proteins modulate the function of ribonucleoprotein (RNP) enzymes through structural changes to their associated catalytic RNA. This goal is highly relevant to public health due to the growing appreciation for the roles of RNPs in tissue complexity and human diseases. In this proposal, we will use RNase P as a model to test our postulate that the versatility of RNPs is due to protein- mediated structural changes in their RNA cores. Although the primary function of RNase P is 5??-maturation of precursor tRNAs, recent findings suggest an expanded functional mission that includes biogenesis of eukaryotic non-coding RNAs. Eukaryotic and archaeal RNase P consist of a catalytic RPR (RNase P RNA) and multiple (4-10) RPPs (RNase P Proteins), unlike the simpler bacterial version (1 RPR + 1 RPP). Because all RPRs are active on their own in vitro, the need for multiple archaeal and eukaryotic RPPs is unclear. We found from step-wise reconstitutions of archaeal RNase P that its assembly intermediates comprising partial suites of five RPPs and the RPR exhibit activity and fidelity of processing in between the RPR alone or the full holo- enzyme (RPR + all RPPs). These findings motivate our central hypothesis that binding of RPPs to specific RPR regions independently and collectively mediates RNA structural changes essential for assembly and catalysis. We will address this hypothesis with two specific aims to delineate structure-function relationships of intermediates en route to assembly of the full RNP: (1) Dissect the structural basis for the distinct roles of archaeal RPPs in aiding RPR catalysis, and (2) map the assembly landscape of archaeal RPPs on the RPR. To study how RPPs guide the RPR towards its functional state, we propose an innovative combination of site- specific and global structural methods coupled to direct functional readouts. In Aim 1, we will probe archaeal RPR structural changes induced by different suites of RPPs at nucleotide resolution using SHAPE-Seq (selective 2??-hydroxyl acylation analyzed by primer extension sequencing), a high throughput method to probe RNA structures. Inferences from SHAPE-Seq, linking structural changes to functional outcomes, will be guided by the RNA-protein contact sites obtained from tethered-nuclease mapping and validated using assays of RPR mutants. In Aim 2, we will survey the hierarchy and cooperation during RNase P assembly with bulk and single molecule fluorescence kinetic studies. RPP-mediated alterations in RPR conformational sampling will be studied using fluorescence resonance energy transfer, and changes in RPR topology will be uncovered with small angle x-ray scattering and native mass spectrometry. Although activity versus fidelity tradeoffs have shaped the adaptive landscape of many enzymes, we expect our work to provide insights into how multiple RPPs allowed archaeal/eukaryotic RNase P to maintain robust cleavage without compromising processing fidelity on a broad range of substrates. This study will contribute to a framework for understanding the mechanistic basis of RNA-protein cooperation in RNPs and how dysfunctioning RNPs lead to disease.

? DESCRIPTION (provided by applicant): Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting humans and agriculture. It has long been thought that nutrient utilization systems of Salmonella would not make effective drug targets because there are simply too many nutrients available to Salmonella in the intestine. However, we have discovered that during growth in the inflamed intestine Salmonella relies heavily on a single nutrient - fructose-asparagine (F-Asn), which is present at high concentrations in human foods. Mutants that cannot acquire F-Asn are severely attenuated suggesting that F-Asn is the primary nutrient utilized by Salmonella during inflammation. No other organism has been reported to synthesize or utilize this compound, although we suspect that a few other pathogens and members of the normal gut microbiota can utilize it. The apparent lack of F-Asn utilization pathways in mammals and most other bacteria suggests a specific and potent therapeutic target for Salmonella. The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). We hypothesize that if Salmonella can initiate inflammation (or enters a gut that is already slightly inflamed), it can begin tetrathionae respiration during F-Asn catabolism and thereby outcompete the normal microbiota, which are doubly compromised by the inflammation and their ability to only ferment (but not respire) F-Asn. We will test this central postulate and build the foundation for two types of therapeutics to block Salmonella acquisition of F-Asn. In our first specific aim, we will investigate the role of a asparaginase (FraE), kinase (FraD) and deglycase (FraB) in F-Asn utilization. Through biochemical characterization of the individual reactions catalyzed by these Fra enzymes and development of high-throughput assays, we expect to facilitate future screens that will identify small molecule inhibitors of these enzymes. We hypothesize that the FraR transcription factor is a repressor. Therefore, preventing its release from the fra operon promoter would also be of therapeutic interest. We propose to determine the natural inducer of FraR and determine the DNA binding sites of FraR in the fra operon. In the second aim, we plan to employ a combination of metagenomics, selective growth in the presence of F-Asn, and bioinformatics to test the idea that in healthy gut communities there are select members of the microbiota that utilize F-Asn and prevent Salmonella from acquiring this nutrient. Finally, we expect our findings on the enzymology and regulation of F-Asn utilization in Salmonella, and possible competing intestinal microbes, to inform our efforts to design new probiotic bacteria that can reduce the severity and duration of Salmonella infection in mice. Overall, our efforts will lead to a better understanding of Salmonella growth in the inflamed intestine and to novel therapeutics.

? DESCRIPTION (provided by applicant): A mutagenesis screen to develop mouse models for neurological diseases led to our recent finding that neurodegeneration could arise from an epistatic mutation affecting transfer RNA (tRNA) biogenesis and function. Loss of function of GTPBPP2, a protein that we showed resolves stalled ribosomes caused by insufficiency of a tRNA, results in a severe phenotype only when present together with a single C50U mutation in a central nervous system (CNS)-specific tRNAArgUCU, n-Tr20. Mutation of this tRNA, which constituted almost 60% of the brain tRNAArgUCU pool, caused a dramatic decrease in processing and thus aminoacylation, and led to ribosomal pausing at AGA codons on cerebellar transcripts. Without GTPBP2 to recycle stalled ribosomes, disease developed with pronounced locomotor defects and neuron loss in the cerebellum beginning at four weeks of age. Neurons also degenerated in other brain regions beginning at 6 weeks of age. While the brain- specificity of phenotypes is consistent with the expression pattern of tRNAArgUCU, there were notable differences in the processing of the C50U tRNAArgUCU depending on the region (cerebellum < cortex, hippocampus; postnatal day 30 < day 0 or 10). Therefore, we postulate that tissue- or development-specific distinctions in the inventory of enzymes that acts on tRNAs are key determinants for disease etiology. It is in this regard that we focus on RNase P, which catalyzes removal of the 5?? leader of pre-tRNAs. Although mammalian liver RNase P functions as a ribonucleoprotein (RNP) consisting of a ribozyme and 10 protein cofactors, there is growing evidence that RNase P may be subject to remodeling especially in the brain. Thus, altered substrate recognition could stem from structural alterations either in the pre-tRNA substrate or in RNase P. These findings provide the framework for our hypotheses: (i) the C50U tRNAArgUCU mutant has a destabilized structure that disables 5?? processing by RNase P, and (ii) spatiotemporal variations in brain RNase P make-up engender defects in processing pre-tRNAs with mutations. We will test these ideas by pursuing two aims. First, we will test the activity of partially purified mouse cerebellar RNase P towards wild-type (WT) and C50U pre- tRNAArgUCU. We will also use chemical/enzymatic probing and high-resolution structural studies to map structural differences between the WT and mutant pre-tRNAArgUCU. Second, we will perform expression analysis and RNP affinity purification to investigate the make-up of brain RNase P from different regions at specific developmental stages. Collectively, our studies will lead to the first biochemical characterization of mammalian brain RNase P and test the novel idea that spatiotemporal variations in the catalytic repertoire that acts on brain tRNAs as well as other non-coding RNAs partly hold the key to delineating how defects in RNA metabolism might result in neurological disorders.

Project Summary Non-typhoidal salmonellosis is one of the most significant food-borne diseases in the U.S. and globally. We recently used high-throughput genetic screening to identify the Salmonella fra locus, whose mutation causes extreme attenuation of fitness in mice. We then determined that the fra locus encodes five genes involved with the uptake and utilization of fructose-asparagine (F-Asn): fraR, fraB, fraD, fraA, and fraE. The fra locus is found only in the non-typhoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium. Thus, targeting the products of this locus in Salmonella with novel antimicrobials is expected to leave the normal microbiota largely intact. Our characterization of the mechanism of attenuation revealed that mutations in fraB cause an accumulation of the FraB substrate ? 6-phosphofructose-asparate (6-P-F-Asp) ? that is toxic to cells. We propose high-throughput screening (HTS) with three different assays to identify small molecule inhibitors of FraB, a deglycase that converts 6-P-F-Asp to aspartate and glucose-6-P (Glc-6-P). One assay utilizes purified FraB enzyme in a spectrophotometric assay, while another is a growth-based assay utilizing a live-attenuated Salmonella and a ?fra control. We tested the biochemical and cell-based assays at the ICCB-Longwood facility at Harvard, and found them to be simple and robust with Z' ?0.9 and ?0.8, respectively. We propose to identify FraB inhibitors using these two assays to screen up to 500,000 compounds at the ICCB-Longwood facility. In the third assay, we will use in silico structure-based virtual screening of ~250,000 compounds from the NCI database. The hits from both of the ICCB-Longwood screens and the computational screens will be tested again at our home institution. A second independent confirmation will utilize a mass spectrometry-based assay to directly measure build-up of 6-P-F-Asp, the substrate of FraB, in live cells. Hits will be characterized further with regard to their IC50, IC90, Ki, and specificity. Computational chemistry will be employed to better understand the chemical profile of FraB inhibitors, and facilitate quantitative structure-activity relationship (QSAR) studies. Moreover, to gain a structural basis for the potency of hits, we will use X-ray crystallography to determine the atomic-resolution structure of FraB with and without select inhibitors. Successful completion of these aims is expected to facilitate hit identification and characterization, key pre-requisites for lead optimization and advancement to a much needed narrow-spectrum therapeutic for non-typhoidal salmonellosis. Narrow-spectrum antibiotics will have two key advantages: (i) limit the side effects caused by disruption of the normal microbiota, and (ii) avoid selecting for antimicrobial resistance among the normal microbiota. We envision a future cocktail of species-specific drugs that could be used to treat cases of human diarrhea without disruption of the healthy microbiota. A drug that ultimately results from the hits identified in this proposal would be one component of this cocktail.