2001 — 2005 |
Graveley, Brenton R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Function of Pre-Mrna Splicing Activators @ University of Connecticut Sch of Med/Dnt
DESCRIPTION (from the application):The long-term goals of this proposal are to understand the detailed mechanism by which splicing enhancers function in humans and other metazoans, and to identify new human splicing regulatory proteins. In many cases, alternative splicing in metazoans is regulated by RNA sequence elements called splicing enhancers, which are typically located within exons downstream of the intron they regulate. Splicing enhancers are recognized by members of' the SR protein family of essential splicing factors. SR proteins contain an N-terminal RNA binding domain and a C-terminal arginine/serine-rich (RS) domain that functions as a splicing activation domain. The mechanism by which enhancer-bound SR proteins function to activate splicing is currently unclear. One model proposes that SR proteins recruit the splicing factor U2AF to the pre-mRNA while other models propose that U2AF binding is unaffected by splicing enhancers. Biochemical methods and an in vivo protein-RNA cross-linking technique will be used to discriminate between these two models for splicing enhancer function I experiments are also described in which the protein sequences that are required for splicing activation domain function are determined. The role of phosphorylation on the function of SR proteins in enhancer-dependent splicing will also be addressed. Moreover, the structure of a splicing activation domain will be determined by a combination of NMR and X-ray crystallographic approaches. Finally, new splicing regulators will be identified in a screen for proteins that can function to activate splicing in mammalian cells. At least 15 percent of all human diseases are the result of mistakes made by the splicing machinery in selecting the correct splice sites. Alternative splicing requires the splicing machinery to choose between the use of alternative splice Sites. Thus understanding how splice sites are selected in alternative splicing is of direct relevance to human health. In addition, at least 35 percent of human genes are alternatively spliced. Our results will increase our understanding of how this important means of gene regulation is controlled.
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0.904 |
2003 — 2006 |
Graveley, Brenton R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alternative Splicing of the Drosophila Dscam Pre-Mrna @ University of Connecticut Sch of Med/Dnt
[unreadable] DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism by which Dscam alternative splicing is mutually exclusive. The Dscam gene encodes an axon guidance receptor that plays an important role in neural development and is the most extensively alternatively spliced gene known to date. The Dscam gene contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33 and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. It has been proposed that each Dscam isoform might interact with a different set of axon guidance cues and that the collection of Dscam isoforms expressed by a cell will be directly involved in guiding neurons to different addresses. It is therefore likely that individual neurons must in some way be programmed to splice the Dscam pre-mRNA in specific ways. Thus understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neural wiring. This proposal is aimed at understanding the mechanisms involved in regulating the alternative splicing of the Dscam exon 4 cluster which contains 12 mutually exclusive exons. First, we will identify RNA sequences involved in the regulation of Dscam alternative splicing and the proteins that bind to these elements. Second, we will determine the mechanism involved in the developmental regulation of exon 4.2 alternative splicing. Third, we will determine the mechanism by which the SR protein B52 and the general splicing factor dU2AF modulate exon 4.4 alternative splicing. Finally, we will determine the mechanistic basis by which alternative splicing of the Dscam exon 4 cluster is mutually exclusive. Together, these experiments will provide significant insight into the mechanisms involved in regulating alternative splicing, the mechanism responsible for mutually exclusive alternative splicing, and the genetic program that determines the specificity of neural wiring in Drosophila. [unreadable] [unreadable]
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0.904 |
2006 — 2009 |
Graveley, Brenton R. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Mechanisms and Regulatory Networks of Alternative Splicing in Drosophila @ University of Connecticut Sch of Med/Dnt
DESCRIPTION (provided by applicant): Intricate gene regulatory networks direct the development of multi-cellular eukaryotes. Although transcription regulation is the best understood mechanism by which these networks are controlled, it has recently become clear that alternative splicing is an equally important regulatory mechanism. Despite the fact that the majority of eukaryotic genes encode pre-mRNAs that are alternatively spliced (at least 65% of human genes), very few splicing regulatory factors have been identified, and the target genes that are controlled by specific splicing regulators are largely unknown. Alternative splicing is primarily thought to be regulated by auxiliary splicing factors - proteins that are not core components of the spliceosome. Auxiliary splicing factors function by binding to the pre-mRNA where they modulate the association of the spliceosome with the regulated splice sites. Recent work from our group and others, however, suggests that components of the general splicing machinery may also play important roles in regulating alternative splicing. This may in part account for the apparent discrepancy between the numbers of known splicing regulators and regulatory targets. The goals of this research proposal are to identify proteins that regulate alternative splicing and their regulatory targets on a genome-wide level, and to determine the biochemical mechanisms by which these proteins regulate alternative splicing using Drosophila melaongaster as a model system. We will first perform microarray experiments to identify alternatively spliced exons that are regulated by the entire complement of RNA binding proteins encoded by the Drosophila genome and to build a model of the splicing regulatory networks in Drosophila. These experiments will be complemented by a combination of biochemical, genetic, genomic, and bioinformatics experiments designed to determine the mechanisms by which both auxiliary and general splicing factors function to control alternative splicing. Together these experiments will provide tremendous insight into the mechanisms of alternative splicing. Given that the proteins involved in regulating alternative splicing in Drosophila and humans are similar, that a number of human diseases are caused by defects in the normal patterns of alternative splicing, and that the human homologs of many of the proteins we will be studying have been implicated in human diseases, it is likely that the discoveries we make will be of direct relevance to human health.
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0.904 |
2007 — 2010 |
Graveley, Brenton R. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alternative Splicing of the Drosophila Dscam Pre-Drna @ University of Connecticut Sch of Med/Dnt
DESCRIPTION (provided by applicant): The long term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism which Dscam alternative splicing is mutually exclusive. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33 and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. Dscam is therefore the most extensively alternatively spliced gene known to date. The Dscam proteins function as axon guidance receptors that play an important role in neural development and function. In addition, Dscam has also been shown to function as immune receptors that help to defend the organism against pathogens. Current evidence suggests that the identity of the isoforms expressed in individual neurons is critical for the proper wiring of the nervous system and in hemocytes is important for pathogen recognition. Thus understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neuronal wiring and pathogen recognition in Drosophila. This proposal is aimed at understanding the mechanisms involved in regulating the alternative splicing of Dscam. First, we will dissect the splicing regulatory program that controls the expression of specific Dscam isoforms. This will involve making a map of the expression pattern of each of the twelve exon 4 variants in the adult brain. Subsequently, we will investigate the role of candidate splicing regulators in controlling splicing of specific exons in individual neurons in the fly. Second, building on an exciting discovery made during the previous funding period, we will determine the requirement and function of competing RNA base- pairing interactions in mutually exclusive splicing of the exon 6 cluster. Finally, we will investigate the mechanism by which proteins ensure the fidelity of exon 6 mutually exclusive splicing. Together, these experiments will provide significant insight into the mechanisms involved in regulating alternative splicing, the mechanisms responsible for mutually exclusive alternative splicing, and the genetic program that determines the specificity of neural wiring and pathogen recognition in Drosophila.
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0.904 |
2011 — 2014 |
Graveley, Brenton R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alternative Splicing of the Drosophilia Dscam Pre-Mrna @ University of Connecticut Sch of Med/Dnt
DESCRIPTION (provided by applicant): The long term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism by which Dscam alternative splicing is mutually exclusive. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33, and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. Dscam is therefore the most extensively alternatively spliced gene known. The Dscam proteins functions as axon guidance receptors that play an important role in neural development and function. In addition, Dscam has been shown to function as immune receptors that help to defend the organism against pathogens. Current evidence suggests that the identity of the isoforms expressed in individual neurons is critical for proper wiring of the nervous system and in hemocytes is important for pathogen recognition. Thus, understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neuronal wiring and pathogen recognition in Drosophila. This proposal is aimed at understanding the mechanisms involved in regulating alternative splicing and the mechanism of mutually exclusive splicing of Dscam. First, we will determine the expression pattern of the exon 4 variants in the nervous system at single cell resolution and explore how regulatory proteins we have identified in tissue culture-based RNAi screens control Dscam splicing in individual neurons in the fly. Second, we will address many important issues regarding the mechanisms of mutually exclusive splicing. Specifically, we will functionally dissect the role of RNA secondary structures in exon 6 mutually exclusive splicing and identify other features such as distance, splice site strength, and HRP36 binding impact exon 6 splicing. Moreover, we will determine the order in which the Dscam introns are removed from the pre-mRNA as this has important implications on the splicing regulatory mechanisms. We will also determine if the splicing of exons within one cluster impacts splicing in other clusters. Finally, we will compare the ability of the insect and vertebrate splicing machinery to process pre-mRNAs containing clusters of three or more mutually exclusive exons. PUBLIC HEALTH RELEVANCE: These experiments will provide tremendous insight into the mechanisms of alternative splicing and mutually exclusive splicing. As the vast majority of human genes are alternatively spliced, it is likely the discoveries we make will be of direct relevance to human health. Moreover, the results will provide significant insight into the mechanisms of neural wiring and immunity.
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0.904 |
2011 — 2014 |
Graveley, Brenton R. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Trans-Splicing in Drosophila @ University of Connecticut Sch of Med/Dnt
DESCRIPTION (provided by applicant): Abstract: Pre-messenger RNA (pre-mRNA) splicing is a process in which introns are removed to join exons together. This process is used during the synthesis of nearly all metazoan mRNAs (~93% of human mRNAs) and is an important means of regulating gene expression. With only a handful of exceptions, splicing occurs in cis - the exons that are joined together are located on the same pre-mRNA. In some eukaryotes, including nematodes, trypanosomes and planarians, splicing can occur in trans. In these cases, a specialized spliced leader RNA is spliced to the 5' end of protein coding RNAs. Interestingly, there are a few cases where a distinct type of trans-splicing has been shown to occur, namely, the splicing of exons from protein coding genes. The two best characterized examples are the mod(mdg4) and lola genes from Drosophila. In each of these genes, a group of common 5' exons can be spliced in trans to one of several (26 for mod(mdg4) or 22 for lola) variable 3' exons. One convincing case of trans-splicing has been shown to occur in mosquitos and one has been recently shown in humans. In each of these cases, trans-splicing of these genes was discovered fortuitously. As a result, the true extent of trans-splicing is unknown. Moreover, nothing is known about the mechanisms involved in trans-splicing. We have recently used deep sequencing to identify 80 new genes that are trans-spliced in Drosophila. The long- term goals of this project are to further explore the trans-splicing landscape in Drosophila and to determine the mechanisms involved in this process. We will first perform an exhaustive survey for trans-spliced genes throughout development in different Drosophila species. Second, we will perform experiments to test whether the trans-spliced mRNAs are translated and are functional. Finally, these experiments will be complemented by a combination of biochemical, genetic, genomic, cell biology, and bioinformatics experiments designed to determine the mechanisms by which trans- splicing occurs. Together these experiments will provide tremendous insight into the mechanisms of trans-splicing, a completely understudied yet important process. Given the potential utility of trans-splicing in treating human diseases and that trans-spliced mRNAs in humans have recently been linked to cancer, it is likely the discoveries we make will be of direct relevance to human health. PUBLIC HEALTH RELEVANCE: These experiments will provide tremendous insight into the mechanisms of trans- splicing. Given the potential utility of trans-splicing in treating human diseases and that trans-spliced mRNAs in humans have recently been linked to cancer, it is likely the discoveries we make will be of direct relevance to human health.
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0.904 |
2012 — 2017 |
Graveley, Brenton R. |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Comprhensive Analysis of Functional Rna Elements Encoded in the Human Genome @ University of Connecticut Sch of Med/Dnt
The goal of this proposal is to comprehensively characterize the functional sequence elements encoded in the human genome that are recognized by 250 RNA binding proteins (RBPs) in two cell lines. To do this, we will generate stable HeLa-S3 and GM 12878 cell lines expressing epitope-tagged RBPs and determine the sub cellular localization pattern of each RBP. These cells will be used to perform CLIP-Seq assays to define genome-wide, and at single-nucleotide resolution, the RNA sequence elements recognized by 250 RBPs. The RNA sequence elements identified will be validated using sequence-based in vitro binding assays. Furthermore, ChlP-Seq will be performed for all nuclear localized RBPs to determine the regions of the genome and chromatin that each RBP associates with. These binding assays will be supplemented with functional assays in RBP-depleted cells that will be critical for assigning functions to the identified binding sites. These assays include RNA-Seq of total cellular RNA and RNA purified from various cellular fractions, ribosomal footprint profiling, and Gro-Seq. Together, these assays will provide functional information regarding the roles of each RBP in splicing, cleavage and polyadenylation, RNA stability, RNA editing, translation, RNA localization, and transcription. Bioinformatic analysis will be performed, largely using software generated by our group, to quantitate all assays and to associate functions to the sequence elements identified in the binding assays. Together, these experiments will provide a comprehensive and in depth measure of the functions of approximately half of the human RBPs and the functional sequence elements that they interact with. This project will fill a major gap in the catalog of functional elements encoded in the human genome that are being characterized by the ENCODE consortium. The product of this project will be a unique and valuable community resource that will push the field forward in new and exciting ways and will almost certainly create new paradigms regarding the functions of RBPs and RNA-protein networks in human biology and disease.
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0.904 |
2016 — 2020 |
Graveley, Brenton R. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Complex Rna Processing @ University of Connecticut Sch of Med/Dnt
? DESCRIPTION (provided by applicant): Most eukaryotic pre-mRNAs, especially in metazoans, are alternatively spliced to generate multiple mRNAs and proteins. Given the importance of alternative splicing in regulating gene expression and enhancing the diversity of the proteome, it is essential to understand the mechanisms of splicing and how alternative splicing is regulated. In this project, we will study three unusual types of splicing in Drosophila The Down Syndrome Cell Adhesion Molecule 1 (Dscam1) gene is the most extensively alternatively spliced gene know. Dscam1 contains 115 exons, 95 of which are alternatively spliced and has the potential to generate 38,016 different mRNA and protein isoforms. We will study how probabilistic splicing of Dscam1 is achieved and how it contributes to determining cell identity, a critical process involved in determining the specificity of neural wiring. The longitudinal lacking (lola) and modifier of mdg4 (mod(mdg4)) genes are the best examples of genes that undergo trans-splicing - a process by which exons from different pre-mRNAs are spliced together to generate a protein-coding mRNAs. We will study how trans-splicing occurs and the role of homologous chromosome pairing in this process. How very long introns are efficiently spliced has long been a mystery. Seventeen years ago, it was shown that one long Drosophila is removed in a progressive, stepwise fashion by a process called recursive splicing. However, until now it was not known how widespread this phenomenon occurred. We recently identified nearly 200 instances of recursive splicing in Drosophila and that it also occurs in humans. We will focus on elucidating the mechanisms and functions of this unusual process. We will also work with collaborators to study how bacteria and archaea become resistant to newly encountered viruses by means of the CRISPR pathway and the determinants and regulators or RNA turnover on a genome-wide scale. All of these problems will be addressed using a wide variety of cutting edge approaches we have applied or developed including reporter genes to facilitate visualizing splicing in single neuron resolution, single cell RNA- Seq nanopore sequencing, RNAi or CRISPR screens, BAC recombineering, Drosophila genetics, and computational genomics. We will also continue to develop additional innovative approaches to address these issues as needed or as opportunities arise due to technical advances in the field.
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0.904 |
2016 — 2017 |
Graveley, Brenton R. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High Throughput Validation of Functional Variants That Affect Pre-Mrna Splicing @ University of Connecticut Sch of Med/Dnt
? DESCRIPTION (provided by applicant): Understanding the functional impact of genomic variants is one of the major goals of modern genetics and the underpinning of personalized medicine. To some extent, it is relatively easy to understand how non-synonymous protein coding variants exert their effects. Many synonymous and non-coding variants are known to act by altering the ef?ciency of pre-mRNA splicing. However, in most cases, it is exceedingly dif?cult to predict how these variants impact pre-mRNA splicing. Thus, a method that could simultaneously measure the splicing ef?ciency of thousands of exons and their variants would have a tremendous impact on the ?eld and our understanding of genome function. We propose to develop an assay we call Variant Exon analysis by Sequencing (VEX-Seq) that will facilitate the simultaneous analysis of the impact of intronic and exonic variants on the splicing of thousands of exons. This approach has the potential to have an extremely high impact on our understanding of genome function and how non-coding sequence variants impact pre-mRNA splicing.
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0.904 |
2017 — 2019 |
Graveley, Brenton R. Pyle, Anna Marie [⬀] Torbett, Bruce Edward (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Monitoring Variation in Mixtures of Long Rnas With End-to-End Rt Sequencing
The ability to sequence cellular RNA molecules has become an essential tool in clinical practice and biomedical research because it provides critical information on gene expression and variation. RNA sequencing technology is made possible by a unique type of enzyme: the reverse transcriptase (RT), which copies RNA molecules into DNA strands that can then be amplified by PCR. Unfortunately, conventional RTs are not highly processive and they only make short copies of RNA templates (short reads), which are then computationally stitched together using a reference genome in order to infer the sequence of intact RNA molecules. During this process, information on the relative prevalence and linkage between distal mutations, RNA editing sites and alternative splice sites within individual transcripts is lost. Many important cellular and viral RNAs are quite long (>1000 nts), and therefore accurate end-to-end sequencing will be required to conduct meaningful studies of their function and diversification. We recently discovered an ultra-processive RT from a eubacterial group II intron (the E.r. RT). Even without extensive optimization, the E.r. RT copies highly structured viral transcripts that are >9kb in size. Our goal is to optimize and enhance the E.r. RT to produce a robust reagent that is widely suitable for diverse biotechnology applications (Aim 1). We will then employ it to address two areas of major unmet need. The E.r. RT will be incorporated into Next Generation Sequencing (NGS) pipelines for monitoring viral evolution and drug resistance in HIV-infected patients (Aim 2). It will then be optimized to generate full-length cDNA libraries from known, but complex mixtures of RNA molecules and then utilized for whole-transcriptome sequencing in order to monitor the tissue specificity of alternative splicing in Drosophila (Aim 3). By performing biochemical optimization in parallel with real-world applications of the E.r. RT, we aim to create a powerful new RT reagent that fundamentally improves NGS, making it possible to study any mixture of RNA transcripts, allowing for genomic phasing and linkage analyses.
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0.903 |
2018 — 2021 |
Graveley, Brenton R. |
U41Activity Code Description: To support biotechnology resources available to all qualified investigators without regard to the scientific disciplines or disease orientations of their research activities or specifically directed to a categorical program area. |
A Comprehensive Functional Map of Human Protein-Rna Interactions @ University of Connecticut Sch of Med/Dnt
PRODUCTION CORE ? PROJECT SUMMARY The objective of the ENCORE (Encyclopedia of RNA Elements) project is to develop a foundational, functional map of protein-RNA interactions of RNA binding proteins (RBPs) encoded in the human genome, and the RNA elements they bind to across the transcriptome. These RNA elements, when expressed, form the basis of co- and post- transcriptional regulation of human genes. Our strategy consists of developing and integrating a physical map of 300 RBPs in two different human cell lines with transcriptome-wide measurements of the effects of depleting these RBPs, their localization patterns and their binding preferences independent of co-factor associations. In this data production core, we will capitalize on the highly efficient data production pipeline we have established over the past four years to generate RNA bind- n-seq, RNA-seq, protein localization and eCLIP data for an additional 300 RBPs. When combined with the data we generated over the past four years, these efforts will culminate in a comprehensive map of the functional RNA elements recognized by essentially all RBPs expressed in two human cell lines, representing approximately half of the known complement of human RBPs. In summary, the data we will produce in this project will enable a more systematic and comprehensive understanding of the role of RBPs and RNA biology in the contribution to human biology and disease.
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0.904 |
2020 — 2021 |
Graveley, Brenton R. Lee, Charles |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
The Uconn/Jax-Gm Training Program in Genomic Science @ University of Connecticut Sch of Med/Dnt
PROJECT SUMMARY We propose to establish the UConn/JAX-GM Training Program in Genomic Sciences to train the next generation of genomic Scientists. This program leverages the unique combination of a recent, but thriving relationship between the University of Connecticut Health Center (UConn Health) and Jackson Laboratory for Genomic Medicine (JAX-GM) in Farmington, CT. Together UConn and JAX-GM have exceeded the critical mass of faculty specializing in genomic sciences to establish Farmington, CT as one of the leading hubs of excellence in genomic research in the world. For example, our research faculty include leaders of the ENCODE, 1000 Genomes, TCGA, 4D Nucleome, and Microbiome consortium projects. Both JAX and UConn also have outstanding histories of excellence in training. Our institutions provide outstanding facilities to perform genomic research including excellent computational resources, easy access to every DNA and RNA sequencing platform, a stem cell and genome engineering core facility, and a joint UConn/JAX-GM single cell genomics facility. We propose to leverage our expertise and truly unique training environment to educate and mentor pre-doctoral trainees in modern genomic sciences. This training will include didactic courses, seminar series, hands on technical workshops, and attending international conferences in genomics. Trainees will be exposed to both wet and dry genomic research and will be able to both generate and analyze genomic data. Trainees will be given a strong foundation in both the underlying biology of the systems being studied and the computational and statistical methods required to correctly interpret data. Trainees will also be educated about ethical scientific behavior.
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0.904 |
2021 |
Graveley, Brenton R. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Genomic Analysis of Nucleic Acid Transactions @ University of Connecticut Sch of Med/Dnt
Project Summary (as submitted in original application) Most eukaryotic pre-mRNAs, especially in metazoans, are alternatively spliced to generate multiple mRNAs and proteins. Given the importance of alternative splicing in regulating gene expression and enhancing the diversity of the proteome, it is essential to understand the mechanisms of splicing and how alternative splicing is regulated. In this project, we will study the roles of RNA binding proteins in alternative splicing, with an emphasis on how RNA binding proteins auto- and cross-regulate the splicing their own and other RNA binding protein genes. This work will provide new insight into the mechanisms of RNA processing and how these proteins regulate one another to achieve homoestasis. Many prokaryotes encode CRISPR-Cas systems which are RNA-guided adaptive immune systems that protects prokaryotic organisms against invaders such as viruses and plasmids. Immune memories are encoded as short DNA sequences, called ?spacers?, that match invader genomes and are stored as interspersed elements in an array of short repeats (the CRISPR array). The CRISPR arrays are transcribed and processed into guide RNAs which pair with Cas nucleases to recognize and degrade target nucleic acid (interference). New immune memories are formed during ?adaptation? when fragments of invader DNA are acquired and integrated into CRISPR arrays for use in future targeting. While a tremendous amount is known about the targeting and degradation of invading nucleic acids, much less is known about the process of adaptation. We plan to further characterize the adaptation process in prokaryotic CRISPR-Cas systems. This work will also provide insight into the mechanisms of adaptation in the immune systems of prokaryotes. In addition to enhancing our understanding of the basic science of prokaryotic immune systems, there is tremendous potential that this work could lead to the development of new tools that can be used for genome editing applications. All of these projects will be addressed using the types of general approaches we have developed such as splicing reporters, single cell RNA-Seq, nanopore sequencing, RNAi or CRISPR screens, and computational genomics. We will also continue to develop additional innovative approaches to address these issues as needed or as opportunities arise due to technical advances in the field.
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0.904 |
2021 |
Graveley, Brenton R. Pyle, Anna Marie [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High-Throughput Detection of Transcriptomic and Epitranscriptomic Variation and Kinetics Using Marathonrt
Project Summary The discovery and characterization of an efficient, ultraprocessive reverse transcriptase (MarathonRT) now makes it possible to develop high-throughput methods for accurate end-to- end sequencing of long RNA transcripts, thereby preserving information content on alternative splicing, editing and modification isoforms while conserving positional linkage information, thereby enabling one to distinguish RNA isoforms in complex mixtures without mapping to a reference genome. This type of technology is essential for deciphering the role of post- transcriptional RNA processing events during control of developmental stage, cell and tissue specificity and regulation of gene expression in higher organisms. It must be sufficiently efficient and accurate to power the long-read sequencing approaches that are used in single- cell RNAseq, particularly when transcript diversification is monitored as a function of time. The first two aims of the proposal are focused on high-throughput detection of RNA modifications (such as 2-O-methyl groups and N7-methyl guanosines). In the first aim, a unique MarathonRT primer extension protocol will be combined with a trained mutational profiling algorithm to recognize the positions and chemical identities of specific RNA modifications, reporting a modification signature that can be recognized at high throughput during long-read sequencing (MRT-ModSeq). In the second aim, MRT-ModSeq will be tested on unknown RNAs, where it will be used to predict sites of modifications on challenging long transcripts and robustness of the predictions will be directly evaluated using mass spectrometry. The second half of the proposal is focused on identification of linked alternative splicing and editing sites on long transcripts within complex cellular mixtures. In aim 3, MarathonRT will be incorporated into a workflow for accurately profiling the relative abundance and processing diversity of the highly complex paralytic (para) gene, which encodes more than 1 million possible processing variants, a subset of which are essential for the voltage-gating of a sodium channel. This sets the stage for Aim 4, in which sensitivity of the workflow must be further optimized and merged with data analysis strategies suitable for time-resolved single cell applications. The resulting method will be tested by monitoring full-length transcriptomic signatures induced by cell stress.
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0.903 |