2001 — 2013 |
Hertel, Klemens J |
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. |
Mechanisms of Enhancer Dependent Splice Site Activation @ University of California Irvine
DESCRIPTION (from the application): Our long term goals are to understand how splice sites for pre-mRNAs are recognized by the spliceosome and to determine how the coupling of pre-mRNA processing with transcription by RNA polymerase II (pol II) contributes to this decision. The general strategy is to characterize pre-mRNA splicing in vitro, as it allows precise control over the reaction conditions. The central hypothesis is that complexes assembled on exonic splicing enhancers (ESE) assist in the recruitment of the spliceosome to adjacent introns, and that the elongation transcription machinery aides this assembly by interacting with splicing factors. Three specific aims are proposed: Understand the mechanisms involved in the positive control of fru pre-mRNA alternative splicing. It is hypothesized that the fru ESE increases U1 snRNP binding to the regulated 5' splice site. We will carry out an in-depth study to analyze the structure and function of a splicing enhancer that controls the activity of the fru female specific 5' splice site. We will examine the role of the fru ESE in spliceosomal assembly, and analyze the positional effects of the dsx/fru ESE complex in activating a 3' or a 5' splice site. Characterize the architecture and assembly of the dsx splicing enhancer complex. It is hypothesized that the assembly of the dsx ESE complex requires highly cooperative interactions. We propose to carry out a detailed structural analysis of the protein complex formed on the dsx ESE. We will determine the function of Tra, Tra2, and RBP1 in the recruitment of the splicing machinery, define the cooperative assembly and stoichiometry of the dsx ESE heterotrimeric complex, map protein-protein interactions within the complex, and characterize mutants in Tra and Tra2. Determine how transcription of pre-mRNAs by polymerase II affects ESE dependent splicing. We have established an in vitro assay to analyze the efficiency of pre-mRNA splicing when coupled to transcription. Using this assay we will test the hypothesis that the level of C-terminal domain (CTD) phosphorylation influences ESE dependent splice site activation and determine at which step during transcription the splicing machinery associates with nascent pre-mRNA.
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2006 — 2009 |
Hertel, Klemens J |
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. |
Mechanisms of Enhancer Dependent Splice-Site Activation @ University of California-Irvine
DESCRIPTION (provided by applicant): Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and pre-mRNAs containing multiple introns and exons can be alternatively spliced in a cell type, cell cycle, or developmentally regulated manner by joining different pairs of 5'and 3'splice sites. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The overall goal of this research proposal is to understand the mechanisms involved in splice-site recognition and pairing of pre-mRNAs. During the previous funding period, we have developed quantitative assays to provide new insights into the mechanisms of splice-site pairing. In the next phase of investigation, we propose to determine the ?molecular events that lock splice sites into a pairing position and to analyze how the combinatorial contribution of multiple splicing signals influence exon inclusion. Specifically, we will determine the biochemical steps that lead to splice-site pairing in A complex (Aim 1). We will test the hypothesis that ATP hydrolysis during A complex formation drives the irreversible juxtaposition of alternative splice sites or exons. In Aim 2 we will determine how the spliceosome executes commitment to splice-site pairing. We will use immuno-depletion and RNAi approaches to test the hypothesis that a subset of U2 snRNP components and associated proteins (CUS2/Tat-SF-1, Prp5, SF3a120, and UAP56) is necessary for irreversible splice-site pairing. Aim 3 describes a systematic and quantitative approach to determine how the probability of exon definition and inclusion is influenced by the combinatorial contributions of variable splice sites, enhancers, silencers, and the exon/intron architecture. We will test the hypothesis that measures of exon inclusion can be quantitated and used to improve the predictability of constitutive and alternative splicing within the human genome. These experiments are important because 1) the commitment to splice-site pairing constitutes arguably the most crucial step during the splicing reaction because it determines the splicing patterns of pre- mRNAs, and because 2) a quantitative framework of combinatorial exon recognition will elucidate mechanisms of splicing regulation and allow to predict the intrinsic pattern of splicing from sequence analysis.
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2007 — 2008 |
Hertel, Klemens J |
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.) |
Genomic Analysis of Alternative Splice-Site Selection @ University of California Irvine
[unreadable] DESCRIPTION (provided by applicant): With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 60% of all human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. The Hertel laboratory (Principal Investigator) has successfully used quantitative approaches to uncover important regulatory aspect of the pre-mRNA splicing reaction. The Baldi laboratory (Collaborator) has been instrumental in the development of bioinformatics approaches required for the analysis of large datasets. In this proposal, the expertise of both groups will be combined to determine the mechanisms of alternative splice-site activation, one of the most frequent forms of alternative pre-mRNA splicing. Biochemical assays will be complemented by computational analyses of large EST databases to provide a molecular understanding for alternative splice-site activation. The long- term goal of this research proposal is to understand the mechanisms by which the splicing machinery correctly identifies exons and faithfully removes intervening sequences. The experiments proposed in this application have two major goals: To characterize the activation of alternative 5' splice sites (Specific Aim 1) and to characterize the activation of alternative 3' splice sites (Specific Aim 2). Preliminary computational analysis of the human genome demonstrated an unusually high frequency of alternative 5' splice-site activation 4 nucleotides upstream or downstream of the dominant 5' splice site. However, it is not clear whether these overlapping splice sites are activated stochastically or whether they are induced by regulatory elements. In Specific Aim 1 we propose biochemical experiments and computational analyses to evaluate these possibilities. Furthermore, we will test the hypothesis that alternative splicing of overlapping 5' splice sites serves to reestablish reading the frame disrupted by additional splicing events. In Specific Aim 2, we will perform a similar set of computational and biochemical experiments to determine why 3' splice-site activation 3 nucleotides upstream or downstream of the dominant splice site is the most frequent form of alternative 3' splice-site usage within the human genome. [unreadable] [unreadable] [unreadable]
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2010 — 2011 |
Hertel, Klemens J |
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.) |
The Role of Alternative Pre-Mrna Splicing in Breast Cancer Progression @ University of California-Irvine
DESCRIPTION (provided by applicant): Recent work has documented that many changes in alternative pre-mRNA splicing associate with breast cancer. These observations raise the question whether changes in pre-mRNA splicing contribute to breast cancer? Another unanswered question is whether breast cancer-specific splicing events are the result of mis-regulations or whether they are the consequence of a general splicing defect in tumors? We have demonstrated that alternative splicing events associated with breast cancer are not caused by changes in the intrinsic ability of the spliceosome to remove introns. Here, we will carry out experiments to gain insights into the question whether alternative splicing contributes to breast cancer progression. In this application, we will characterize pre-mRNA splicing networks to test the hypothesis that aberrant expression of splicing regulators trigger breast cancer-specific alternative splicing. We will combine experimental analyses with bioinformatics to characterize breast cancer-specific alternative splicing. (1) Using high throughput sequencing approaches and computational analyses of EST databases we will generate and validate a comprehensive list of breast cancer-specific alternative splicing events. (2) Using real-time PCR and quantitative western blot analysis, we will test the hypothesis that splicing regulators are differentially expressed in breast cancer. We will combine these expression profiles with computational analyses of breast cancer-specific alternative splicing events and pre-mRNA splicing predictions to determine likely targets of splicing regulatory networks. The approaches taken integrate high-throughput data generation with bioinformatics and classical molecular biology methods to gain important new insights into splicing regulatory networks. The proposed experiments take advantage of the most up-to-date experimental and analytical approaches to generate a snapshot of gene expression unique to breast cancer, thus producing a wealth of data useful to the breast cancer research community. The described experiments will demonstrate whether alternative pre-mRNA splicing is a significant contributor to breast cancer biology. Introducing these molecular insights to analyses of tissues obtained from breast cancer patients of various stage groupings will indicate to what degree changes in alternative splicing contribute to breast cancer progression, thus, providing additional screening and outcome prediction possibilities. PUBLIC HEALTH RELEVANCE: Pre-mRNA splice variants have been identified for a large variety of breast cancer genes, suggesting that widespread aberrant and alternative splicing may be a consequence or even a cause of breast cancer. Here, we propose to evaluate to what degree alternative pre-mRNA splicing contributes to breast cancer progression. The value of the molecular insights gained is realized when applying such analyses to breast cancer patients of various stage groupings, as they are likely to provide additional screening and outcome prediction possibilities.
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2014 — 2018 |
Edwards, Robert Andrew Hertel, Klemens J Shi, Yongsheng Waterman, Marian L (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. |
Coordinated Regulation of Alternative Pre-Mrna Processing in Colon Cancer @ University of California-Irvine
DESCRIPTION (provided by applicant): Recent work has documented that change in alternative pre-mRNA processing associate with colon cancer. These observations suggest that aberrant pre-mRNA processing may contribute to tumorigenesis. We have demonstrated that the expression levels of several RNA processing factors are misregulated in colon cancer and that generation of alternative mRNA isoforms of the Wnt-signaling LEF/TCF transcription factors activates cell growth. These observations strongly suggest that alternative pre-mRNA processing participates in establishing altered gene expression programs that are characteristic for colon cancer. Here we propose to determine the extent of alternative pre- mRNA processing in colon cancer and to test whether colon cancer-specific mRNA isoforms provide a functional advantage for tumor growth and progression. We will combine experimental analyses with bioinformatics to characterize colon cancer-specific alternative pre-mRNA processing (splicing and polyadenylation). (1) Using high throughput sequencing approaches and computational analyses we will generate a comprehensive list of colon cancer-specific alternative pre-mRNA processing events. (2) Using mRNA isoform-specific knockdown and overexpression approaches we will test the hypothesis that the misexpression of mRNA isoforms changes the colon cancer cell phenotype and alters the ability of normal colon to maintain proper cell cycle control. We will also test the hypothesis that the altered expression of candidate mRNA isoforms triggers tumor formation using colon cancer xenograft models in nude mice. (3) Using cell-based assays and xenograft models we will test the hypothesis that misregulated pre-mRNA processing factors influence proteomic diversity and colon tumorigenesis. The experiments described will highlight colon cancer-specific alternative pre-mRNA processing events that may be crucial for the development of additional screening and outcome predictions. Furthermore, understanding the mechanisms involved in colon cancer-specific alternative pre-mRNA processing may pave the way for novel therapies of human colon cancer. Throughout this project we will rely on our expertise in cancer biology, RNA biology, and bioinformatics to obtain snapshots of gene expression patterns in colon cancer, thus producing a wealth of data useful to the colon cancer research community. The identification of colon cancer-specific alternative pre-mRNA processing events coupled with differential gene expression patterns will be valuable for early detection and improved prediction of colon cancer prognosis. Ultimately, the alternative pre-mRNA processing analysis may uncover new targets for colon cancer therapy.
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2015 — 2018 |
Hertel, Klemens J |
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. |
Tracking Gene Expression Dynamics From Transcription to Degradation @ University of California-Irvine
? DESCRIPTION (provided by applicant): The regulation of gene expression dictates when, where, and to what degree protein isoforms are generated, thereby meeting the metabolic needs of various cell types that make up organs and living beings. Given the constantly changing microenvironment, this process is highly dynamic, capable of adjusting instantly to multiple external cues, or mounting gene expression responses within extended periods of time. To achieve diverse gene expression responses cells rely on multiple points of regulation that include modulating the origin and rate of transcription, altering pre-mRNA processing to induce the generation of alternatively spliced mRNA isoforms, changing the length or location of the 3' UTR through alternative polyadenylation, selective mRNA isoform translation, and altering the stability of mRNA pools. All of these gene expression steps are integrated and co-dependent. However, our understanding of the dynamic nature of gene expression is limited because most cell-based investigations analyze only steady-state levels of gene expression and because detailed analyses focus only on one of the multiple steps involved in representing a gene expression profile. This research application focuses on evaluating the dynamics of gene expression from pre-mRNA generation through RNA processing in the nucleus to translation and mRNA degradation in the cytoplasm with the long-term goal to chart the life span of all expressed mRNAs upon cellular transformation. We will test the hypothesis that alterations in the kinetics of gene expression steps set in stone unique gene expression programs that dictate cellular fate. Using novel metabolic labeling techniques we will track pre-mRNA dynamics in the nucleus (Aim 1) and mRNA dynamics in the cytoplasm (Aim 2) to develop a comprehensive understanding on the kinetic interplay that establishes steady-state gene expression patterns. Aim 3 will use this understanding and tools to determine to what degree alterations in the dynamics of gene expression steps contribute to the overall change in gene expression profiles that accompany Src-induced breast epithelial cell transformation. The experiments described will highlight how crucial the interplay between transcription, RNA processing and mRNA translation is for the development of altered gene expression programs. Furthermore, understanding the kinetics involved in establishing novel gene expression profiles may pave the way for novel therapies of human breast cancer. Throughout this project we will rely on our expertise in kinetics, RNA biology and bioinformatics to obtain complete kinetic profiles of gene expression unique to Src-induced breast epithelial cell transformation, thus producing a wealth of data useful to the gene expression and breast cancer research communities. The identification of breast cancer-specific gene expression events may be valuable for the development of novel early detection or prediction tools.
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2016 — 2019 |
Hertel, Klemens J |
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. |
Mechanisms of Enhancer Dependent Splicesite Activation @ University of California-Irvine
Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how their combinatorial contribution mediates exon inclusion or exclusion. Complicating the task of experimentally deciphering alternative splicing decisions is the fact that most human genes contain multiple introns and exons that often exhibit more complex splicing patterns than simply selecting between two competing splice sites. This renewal application focuses on understanding the mechanisms of regulated splice-site selection with the long-term goal to predict alternative splicing based on sequence analysis. The experiments outlined below build on the most exciting discoveries made during the previous funding period. Historically, SR proteins have been associated with splicing activation, whereas hnRNPs are known for their inhibition of the splicing reaction. However, new genome-wide analyses suggested that hnRNP-like splicing factors could also activate exon inclusion. We demonstrated that both classes of splicing regulators have the ability to promote or repress splicing, antagonistic activities that simply depend on whether the splicing regulator binds within the exon or within the intron. Thus, SR protein and hnRNPs are functionally interchangeable and their regulation of splicing is dependent on the location of their binding site relative to a splice site. How is it possible that a splicing factor can activate or repress spliceosome assembly? We propose to carry out complementing sets of experiments to determine the molecular mechanisms that switch splicing regulatory proteins from splicing activators to splicing repressors (Specific Aims 1 and 2). In Specific Aim 3 we will determine the frequency position-dependent splicing in living cells and use the new molecular insights to improve splicing predictions.
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