Susan A. Gerbi - US grants

It is the aim of this research to characterize nucleotide sequences in ribosomal RNA (rRNA) both in terms of spatial and functional relationships. Knowledge of the role of rRNA within the ribosome is a prerequisite for complete understanding of protein synthesis, a process common to all degrees of complexity and development. It is necessary to fully understand basic life processes such as these so that we may see how they are altered in pathological and neoplastic cases. We have shown that regions of rRNA are conserved during evolution by methods of RNN-DNA hybridization and comparative fingerprint analysis. We are further characterizing these regions in terms of their primary structural location (by electronic microscopy of spread nucleic acids and by restriction enzyme (mapping), their secondary structure (by gel electrophoresis isolation of hairpin loops), and their tertiary structure in the ribosome.

Our main goal is to define the ORI (origin of DNA replication) sequences needed for DNA puffs II/9A and II/2B in the fungus fly Sciara coprophilia, and to understand the relationship between ecdysone induced initiation of amplification and transcriptional enhancers. DNA puff ORI will be mapped by chromosome walks across II/9A and II/2B coupled with quantitative Southern blots to map the region of maximum amplification for each puff. A newly developed 2-D gel method will be applied to each ORI domain for finer resolution mapping; this method will also show if the same ORI sites are used for prepuff replication (prior to amplification). The DNA sequence of ORI at both puffs will be determined and compared to one another Transcription units active during DNA puffing will be mapped by Northern blots with probes spanning the chromosome walks at both DNA puff loci, and their 5' start sites determined by primer extension. Transcriptional enhancers will be located by P element transformation in Drosophila using CAT assays. DNase I hypersensitivity and in vivo DMS footprinting of enhancer regions will locate binding sites for trans-acting proteins, and these areas will be sequenced. Do enhancers overlap ORI? Future studies will address whether the activated ecdysone receptor directly binds to upstream sites to initiate both amplification and transcription. Gene amplification is an important feature of some cancers, and DNA puffs provide an excellent model system to understand the mechanism of amplification and its hormonal control.

We propose to continue our studies on eukaryotic DNA replication, using DNA puffs from polytene chromosomes of the fly. Sciara coprophila, as a model system. DNA puffs are one of only two cases known to undergo intrachromosomal DNA amplification as a normal event in development (the other case is chorion gene amplification In Drosophila follicle cells). We have just mapped the origin for amplification to predominantly a l kb region In DNA puff 11/9A. Thus, it now provides the only non-viral, metazoan eukaryotic origin that has been mapped unambiguously to a small, well-defined region. We propose to continue our mapping studies to confirm this map location based on 2-D gels by using another method. We will complete our preliminary experiments that suggest that the same region is used as an origin for normal chromosomaI replication prior to amplification. We propose to develop techniques to map the origIn of replication to the nucleotide level; this technique will be developed using a well-defined ARS in yeast before application to Sciara DNA puffs. We will isolate and characterize genomic clones from another large DNA puff (11/2B). and map its origin. The origin regions in DNA puffs 11/9A and 1l/2B will be sequenced to allow comparison between them for Identification of conserved regions that could be of functional importance for replication. DNase I hypersensitivity studies will be completed, and in vivo and in vitro footprinting carried out to define areas that bind proteins in the origin region. The Importance of DNA sequences that function as the origin and the cis-acting elements that regulate origin activity will be tested by functional assays using P-element transformation. Cancer may be thought of as a disease of runaway DNA replication. so an understanding of the fundamental mechanism of DNA replication in eukaryotes and Its normal replication is key to future progress in cancer where the controls have seemingly gone awry.

We propose to continue our studies on eukaryotic DNA replication, using DNA puffs from polytene chromosomes of the fly. Sciara coprophila, as a model system. DNA puffs are one of only two cases known to undergo intrachromosomal DNA amplification as a normal event in development (the other case is chorion gene amplification In Drosophila follicle cells). We have just mapped the origin for amplification to predominantly a l kb region In DNA puff 11/9A. Thus, it now provides the only non-viral, metazoan eukaryotic origin that has been mapped unambiguously to a small, well-defined region. We propose to continue our mapping studies to confirm this map location based on 2-D gels by using another method. We will complete our preliminary experiments that suggest that the same region is used as an origin for normal chromosomaI replication prior to amplification. We propose to develop techniques to map the origIn of replication to the nucleotide level; this technique will be developed using a well-defined ARS in yeast before application to Sciara DNA puffs. We will isolate and characterize genomic clones from another large DNA puff (11/2B). and map its origin. The origin regions in DNA puffs 11/9A and 1l/2B will be sequenced to allow comparison between them for Identification of conserved regions that could be of functional importance for replication. DNase I hypersensitivity studies will be completed, and in vivo and in vitro footprinting carried out to define areas that bind proteins in the origin region. The Importance of DNA sequences that function as the origin and the cis-acting elements that regulate origin activity will be tested by functional assays using P-element transformation. Cancer may be thought of as a disease of runaway DNA replication. so an understanding of the fundamental mechanism of DNA replication in eukaryotes and Its normal replication is key to future progress in cancer where the controls have seemingly gone awry.

DESCRIPTION(applicant's abstract): The production of mature ribosomal RNA (rRNA) species from the- rRNA precursor requires an ordered sequence of cleavage events. The main subject of this grant is to understand the roles of small nucleolar RNAs (snoRNAs) in eukaryotic rRNA processing. Most of the proposed experiments focus on U3 snoRNA, which is the most abundant of the snoRNAs and is essential for cleavage at several sites in prerRNA. Our recent dissection of U3 has identified functionally important areas in the molecule. The studies described here will explore interactions of U3 with pre-rRNA. The Xenopus oocyte will be used as an "in vivo test tube" to deduce rRNA processing mechanisms. Yeast will be used for a genetic screen. (1) Analysis of point mutations in the 5' region of U3 snoRNA will be completed; pseudouridine (w) formation at nt 8 and 12 in Xenopvs U3 snoRNA will be confirmed for endogenous U3 and analyzed for injected U3 mutants. (2) Our recent results suggest a set of dynamic base-pair interactions between U3 snoRNA and prerRNA. These will be assessed by compensatory changes in yeast and in Xenopus. (3) Yeast suppressor screens will be carried out to identify intermolecular contacts of U3 Box A' with RNA and protein. (4) Site-directed mutagenesis of Xenopus pre-rRNA will reveal the sequences and structures required for cleavage at sites AO, I and 2. (5) snoRNAs may modulate the conformation of rRNA during its biogenesis. Chemical modification or pre-rRNAs after depletion of U3 snoRNA or after depletion and subsequent injection of mutant U3 snoRNAs will be used to analyze if U3 snoRNA influences the conformation of pre-RNA. (6) Antisense oligonucleotide depletion in Xenopus oocytes will demonstrate if Ui 3 and 7-2IMRP snoRNAs are required for cleavage at sites 2 and 3, respectively. The proposed studies should elucidate the importance of dynamic RNA-RNA interactions during ribosome biogenesis, a fundamental process essential for cell growth and viability.

The ribosome is a cellular organelle that is required for protein synthesis and is essential for life in all kingdoms. It is composed of structural RNAs (ribosomal RNAs - rRNAs) complexed with a set of ribosomal proteins. Because of the integral role of the ribosome in basic cellular mechanisms, it is important to understand how this crucial molecular machine functions. Recent work has identified conserved nucleotide elements (CNEs) within rRNAs that are perfectly conserved in all eukaryotic rRNAs but not in those from bacteria. CNEs are likely to carry out important eukaryotic-specific functions for the ribosome, such as nuclear export. Preliminary data support the idea that CNE-1 contains nuclear export information, which may be mediated by the proteins that are bound there. This project has two specific aims. The first aim focuses on characterization of CNEs in 28S rRNA and their role in mediating ribosome export. Nuclear export will be assayed for yeast ribosomes that are mutated in CNE-1; these mutant ribosomes will contain an MS2 coat protein (cp) binding site that will bind a green fluorescent protein (GFP)-cp fusion protein to facilitate visualization of the export process. The eight CNEs in large subunit (LSU) rRNA will be screened for nuclear export information, using GFP for live cell imaging in budding yeast. The data will indicate which CNEs in addition to CNE-1 contain sufficient information for nuclear export. Leptomycin B (LMB) inhibition in yeast will test Crm1/Xpo1 as the transport receptor for nuclear export of the CNE chimera. The second aim of the project focuses on CNE binding proteins, which will be identified by three complementary approaches. One involves streptavidin-bead pull-down of biotinylated CNE after incubation with yeast extracts, with subsequent identification of the bound proteins by gel electrophoresis and mass spectrometry. In a second approach, a yeast three-hybrid screen will identify genes which encode proteins that interact with CNE-1. As a third approach, a high copy suppressor screen of rDNA with mutated CNE-1 will be used to identify plasmids with genes that are high copy suppressors. In any remaining time of the three year project period, experiments will be initiated to explore the function of proteins that bind to CNE-1.

The intellectual significance of this project is the elucidation of a nuclear export function for a eukaryotic-specific conserved nucleotide element. The project also has broader significance for science and society. It will advance discovery and understanding while promoting teaching, training and learning, by involvement of undergraduates in the research. It will broaden participation by underrepresented groups, as some of the undergraduates working on this project will be women and others will be minority students from Brown or from minority-serving institutions that participate in the Leadership Alliance which is housed at Brown. The data from these experiments will be published in widely read journals so that it is readily accessible to the scientific community.

Intellectual Merit: The objective of this research is to identify conserved nucleotide elements (CNEs) in ribosomal RNA (rRNA) and elucidate their functions in the ribosome. The CNE regions must be crucial for ribosome biogenesis, structure and/or function as they have been maintained throughout evolutionary time. Presumably, mutations in these regions would have been lethal to the organism and hence not perpetuated. This research project builds upon previous NSF-funded research in which a Complete Organismal rRNA Database (CORD) was developed and bioinformatic methods were designed to identify CNEs in the large ribosomal subunit rRNA. These methods were applied to eukaryotic 25-28S rRNAs and 42 CNEs were thus identified. Of these 42 CNEs, five are universally conserved in all Domains of life (eukaryotes, bacteria and archaea), and nine other CNEs are specific to eukaryotes with their sequences being degenerate in bacteria. This analysis will be expanded to identify CNEs in bacterial 23S rRNA and to discern which bacterial CNEs are universally conserved in all domains of life and which are specific to bacteria. Mutations will be made in each of the nine eukaryotic-specific CNEs to analyze their function in rRNA processing, ribosome export, ribosome function in protein synthesis and coordination with the cell cycle. Furthermore, an MS2-based pulldown strategy will be developed for isolation of ribosomes containing mutations in the rRNA. Development of this system will provide the foundation for future proteomic and structural studies on ribosome biogenesis. The results of the research described here will be of great value to the scientific community. rRNA is used as a yardstick for phylogenetic comparisons, so the derivation of CORD will be useful to evolutionary biologists. Moreover, the results will be very valuable to molecular biologists in their study of ribosomes. The next frontiers of ribosome research are (i) a study of eukaryotic ribosomes, (ii) a full description and understanding of ribosome biogenesis and (iii) elucidation of conformational changes that occur in ribosomes during elongation in protein synthesis. The data from the research described here will highlight the regions in rRNA of great functional importance for these processes, thus helping to focus studies on structure and function by the scientific community.

Broader Impacts: The research described here will be integrated with education, serving to train undergraduates through a postdoctoral associate who will help with this research. Graduate education and postdoctoral training are topics of great interest to the Principal Investigator who has played leadership roles at the national level in this area. The research described here will help to broaden participation in science by women and minorities, with students from these groups working on the research. Furthering the opportunities in science for women and minorities is a topic in which the Principal Investigator has been actively involved at Brown University and at the national level. New methods and tools will be developed by the research described here, thus enhancing the infrastructure for scientific research. The results will be disseminated to the scientific community through publications and talks. The Principal Investigator has a track record of bringing the benefits of biological research to the attention of the lay public and to Congress.

Researchers previously developed a structurally aligned ribosomal RNA (rRNA) database referred to as the Full-Length Organismal rRNA Alignment (FLORA) database. Building on that database the research team plans to further investigate a computational approach for analysis of the FLORA database to contain full length rRNA sequences from each domain of life. This analysis identifies conserved nucleotide elements (CNEs) in rRNA from each domain of life. Also, this analysis identifies the CNEs that are universally conserved in all domains and those that are domain specific (highly conserved in one domain but degenerate in the other two domains of life).

Analysis of the database may assist with identifying new sites in rRNA as targets for the development of new antibiotics for use in humans, animals and plants. The computational approach presented here to identify rRNA targets for new antibiotics is a creative new way to approach antibiotic design and has the potential to be transformative to the field of drug development.

DESCRIPTION (provided by applicant): We propose to identify features in the human genome that define all potential origins (ORIs) of DNA replication and features that define which subset of all potential ORIs will be activated for initiation of DNA replication. The disagreement in prio results by others to map ORIs genome-wide may reflect contamination in nascent strand preparations that were enriched by lambda exonuclease (Lexo). Our preliminary results reveal that Lexo cannot digest G quadruplex (G4) structures; therefore, G4 will significantly contaminate nascent strand preparations. We will correct for this problem by normalization to a matched internal control and by use of buffer conditions that minimize Lexo biases. These corrections will be used in nascent strand sequencing (NS-seq) to map active ORIs genome-wide. Moreover, identification of the strand switch for leading strand synthesis will confirm the position of ORIs of bi-directional replication. We will first apply our revised NS-seq protocol to budding yeast where all ORIs are known in the genome, thus validating our revised NS-seq approach. Next, we will apply the revised NS-seq protocol to the genomes of three human cells lines, allowing us to compare active ORIs used by normal cells (GM06990) to those used in cancer cells (HeLa and MCF7). We will validate our results by use of the orthogonal approach of DNA combing for a few selected ORIs from our data set. DNA combing will also allow us to determine the spacing between active ORIs on single DNA molecules. Chromatin immunoprecipitation (ChIP) will be used to identify all potential ORIs in the genome (those that bind ORC2) and the subset that are active ORIs (those that bind Cdc45). For this, we will employ ChIP-reChIP approaches coupled with ChIP-exo. The Cdc45 binding sites at active ORIs will also serve to validate the active ORIs mapped by NS-seq. The significance of our experiments is two-fold. First, at the biological level, the sum of our results will allow correlatons to be drawn for features in the genome that correlate with the definition of all potential ORIs and the features that correlate with the subset of these that become active ORIs. Second, at the technical level, our corrections for Lexo biases will allow our development of revised protocols for NS-seq, thereby impacting the replication field.

The projected experiments will lead to new advances in understanding of DNA replication and in technology development. DNA replication starts at "origins of replication" of which there are many in all the chromosomes in a cell. There are exquisite controls to ensure that each origin of replication is not activated more than once per cell cycle. When cells override these regulatory controls, regions can become re-replicated leading to gene amplification that is a hallmark of many cancers. To understand such overrides, it is important to have fundamental knowledge about the origins of replication themselves. Although several methods exist to map replication origins, their results are not in full agreement with one another. The focus of this project is to refine a new nanopore sequencing technology using the MinION sequencing device. One future application of the projected refinement is to provide a new method to map origins of replication genome-wide. There are also many other important applications as summarized below.

The project aims to advance the nascent technology of nanopore DNA sequencing to obtain ultra-long reads, using the MinION sequencing device from Oxford Nanopore Technologies. The projected experiments include: (i) improved preparation of ultra-long DNA; (ii) characterizing the signal space of the nanopore device; (iii) testing hybrid assembly methods using the MinION coupled to other methods; (iv) direct nanopore sequencing of non-amplified, single-molecule, ultra-long DNA with modified nucleotides as a platform for future experiments in DNA replication and other areas. The impact of sequencing ultra-long DNA molecules and being able to identify modified, labeled, or damaged bases is enormous and will be transformative to many scientific subfields. Development of methodology to obtain ultra-long MinION reads (up to megabase lengths) will dramatically enhance the contiguity of hybrid and non-hybrid genome assemblies, and will allow the exploration of numerous biological questions with a single molecule perspective. Ultra-long MinION reads as well as signal space operations will also allow researchers to discover the covariation of single nucleotide polymorphism (snp), chromosomal rearrangements, and base modification co variations on single molecules. There will be broad educational impact of the research, both in classroom teaching and as laboratory training for undergraduate and graduate students involved in the project. STEM education will be advanced by lectures in genomics and a Genomics Club organized by the graduate student funded by the grant. The PI will continue her track record to broaden participation by women and underrepresented minorities, and education of the public on biological research.

Site-specific intra-chromosomal DNA amplification is common in many cancers, but the events that initiate this process are unknown. To address this question, we will use one of only two known systems where this occurs as a normal process in development, namely in the salivary gland polytene chromosome DNA puffs of the fly Sciara. We propose to elucidate the mechanism for induction of DNA puff amplification, making use of our recent advances in development of a method for Sciara transformation, assembly of the Sciara genome, and identification of DNA puffs in the genome sequence. First we will map the origins of replication and re-replication in the salivary gland genome, using NS-seq and ORC-ChIP as well as validation of the re-replication origins by DNA combing. Next, we will identify consensus motifs that map near the amplification origins and test them by deletion mutagenesis as well as a functional test by placement at an origin that normally does not re-replicate to ask if it is now driven to re-replicate. With the cis-regulatory elements in hand, we will identify the trans-acting factors (protein and RNA) that bind to these elements by pull-down experiments with in vivo occupancy validation by ChIP. For a functional test, we will inducibly tether the trans-acting factor to an origin that normally does not re-replicate to ask if it is now driven to re-replicate. This will include development of methodology for induction of tethered RNA, if the trans-acting factor is a ncRNA. In the final section of this grant application, we will use the trans-acting factor for pull-down experiments to identify proteins that interact with it. Among many possibilities, such interacting proteins may be components of the replication machinery or modify it to be continually load and activate Mcms or modify the chromatin environment. Our results will suggest a mechanism for DNA puff amplification, and future experiments can test if this may serve as a paradigm for initiation of DNA amplification in cancer.