Claire L. Moore - US grants

The process of polyadenylation is essential in the biogenesis of eukaryotic mRNA. The poly(A) tall is thought to participate in mRNA translation and stability. Defects in the formation of the poly(A) tail decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. In addition, polyadenylation can play a role in the regulation of gene expression, especially in cases with alternative selection of poly(A) sites. In this way, it becomes part of a cell's response to stimuli governing growth, differentiation, and tissue-specific gene expression. The goals of this proposal are to determine the factors responsible for this processing and how they interact with each other to give an active processing complex. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological. state of the cell changes, and how this regulation affects mRNA levels globally or specifically. This research will characterize the factors which recognize the signal sequences on polyadenylation precursor. It will focus on two proteins, pl55 and p68, which require the AAUAAA sequence to bind to precursor RNA, are found in polyadenylation-specific complexes, and dissociate from the RNA once it is cleaved and polyadenylated. These observations are consistent with a role for these proteins in the polyadenylation process, possibly in signal recognition and assembly of the processing complex. Their relationship to the enzymes responsible for cleavage and poly(A) addition will be determined by whether they chromatographically cofractionate. Other experiments using an in vitro system will explore how polyadenylation specific factors interact with each other and with precursor RNA and products during the polyadenylation reaction. Three approaches will be taken to clone polyadenylation-specific factors: a) screening of lambda gt11 human cDNA expression libraries with RNA probes containing polyadenylation signal sequences; b) use of ultraviolet-crosslinked ribonucleoprotein complexes as immunogens to produce antibodies to polyadenylation-specific proteins; and c) sufficient purification of these proteins so as to use them either as antigens for specific antibody production or to obtain protein sequence to generate DNA probes. Either reagent would then be used to screen expression libraries. Using an in vitro system, the 3' end processing of mRNAs from bovine leukemia virus, an oncogenic retravirus, and the role of viral specific factors in this processing will be examined. Finally, the role of polyadenylation in transcription termination will be investigated. Transcriptional templates which encode a self-cleaving RNA sequence and a termination site will be used to determine the role of cleavage of the nascent transcript on termination of RNA polymerase 11 transcription. This will be studied in vivo using transient expression assays, and if possible, with an in vitro system capable of transcription initiation and elongation, polyadenylation, and termination.

The formation of polyadenylated 3' termini is essential to the biogenesis of eukaryotic mRNA. Defects in this processing decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. Regulation at the level of polyadenylation can affect the amount and type of mRNA synthesized from a transcriptional unit. In this way, it becomes part of a cell's response to external stimuli governing growth, differentiation, and tissue-specific gene expression. For these reasons, it is important to understand the mechanism and regulation of polyadenylation. In mammals, maturation of the mRNA 3' end involves cleavage of precursor and addition of adenylate residues to the new end. This processing has been examined in vivo and in cell-free systems. This research has defined how precursor RNA is cleaved and polyadenylated and what signal sequences on precursor direct the processing. Further progress in characterizing the processing activities is severely limited by the lack of suitable molecular genetics in mammalian systems. However, little is known about 3' end formation in eukaryotes such as the yeast S. cerevisiae, which is more amenable to genetic analysis. The primary goal of this research is a thorough molecular analysis of polyadenylation in yeast using a combination of genetics and biochemistry. The results of these studies will allow us to compare the processing mechanism in yeast to that used in metazoans. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological state of the cell changes, and how this regulation affects mRNA level globally or specifically. The aims of this research are: I. Development of a genetic screen to identify the cis-acting RNA sequences and the trans-acting factors necessary for polyadenylation in yeast. Using primary a colorimetric assay, it should be possible to determine the minimal sequences needed for polyadenylation and to screen for mutants which do not recognize a functional polyadenylation signal. The mutant genes can then be identified and cloned by complementary transformation with wild type genes. II. Clarification of the role transcription termination in the formation of the mature 3' end of yeast mRNA. This experiment will use a nuclear run-on assay to detect any transcription beyond the poly(A) addition site. III. Development of an in vitro system in yeast which correctly polyadenylates precursor RNA. The goal of these experiments is to determine the molecular pathway of polyadenylation in yeast, to complement the genetic analysis of signal sequences and trans- acting factors, and to provide an assay for the purification of processing activities from crude extracts.

The formation of mRNA 3' ends involves precise cleavage of precursor followed by addition of adenylate residues to the new end of the RNA. The primary goal of this research is a thorough molecular analysis of polyadenylation in the yeast S. cerevisiae, using a combination of genetic and biochemical approaches. An understanding of the mechanism of polyadenylation will provide the basis from which to ask questions about how the process is regulated as the physiological state of the cell changes, how this regulation would affect mRNA levels globally or specifically, and how polyadenylation interacts with other processes involved in mRNA synthesis. The first specific aim is to biochemically characterize factors involved in yeast rnRNA polyadenylation and to understand at a molecular level how these factors interact with each other and with the precursor RNA. These factors will be purified to homogeneity, using in vitro assays for each step in the processing reaction Peptide sequence or monoclonal antibodies derived from purified components will be used to clone the genes encoding these factors. The second specific aim is to use genetic analysis to study trans-acting factors necessary for polyadenylation. The following screen will be used to detect mutations in processing factors. Yeast strains which are temperature sensitive for growth will be screened for conditional phenotypes in three types of assays: i) production of blue colonies due to transcriptional read-through into beta-galactosidase coding sequences, 2) Northern analysis of RNAs made in vivo from a construct designed to give a discrete poly(A)- read-through product whose end is specified by snRNA termination signals, and 3) examination of extracts made from the best candidates to identify ones which are reproducibly defective for processing in vitro. The genes can be cloned by complementation of the conditional defect with a wild-type yeast genomic library. The genetic analysis will aid the biochemical characterization in several important ways. If one of the factors cannot be cloned by protein sequence or antibody screening, it may be possible to identify it with a genetic screen. Gene disruption will indicate if the proteins identified biochemically are essential for viability in yeast. Finally, genetic strategies such as suppressor analysis, identification of pairs of mutant genes which exhibit synthetic lethality, and directed mutagenesis of the genes, can be used to determine how these factors interact in vivo, and to assign functions to different domains of the proteins. The final specific aim is to further define the signals which specify polyadenylation in yeast. Random PcR and chemical mutagenesis will be used to determine what sequences in addition to the (UA)6 repeat are essential for GAL7 polyadenylation. Debilitating mutations will be detected in vivo using a beta-galactosidase reporter construct, and then tested in vitro for their effects on cleavage and/or poly(A) addition.

Polyadenylation is essential for mRNA synthesis and its efficient translation. Regulation of this process can also affect the amount and type of mRNA derived from a gene, and thus becomes part of the cell's repertoire of responses to stimuli governing growth and differentiation. The enzyme poly(A) polymerase (PAP) is responsible for adding adenosines to the mRNA 3' end. For this activity, it needs binding sites to bring its substrates, ATP and an RNA strand, into proximity at its catalytic center. PAP must also have domains to mediate association with specificity factors which guide it to the appropriate 3' ends and modulate its activity so that it processively synthesizes tails of correct length. The mammalian PAP is further regulated by post-translational modification. The purpose of this study is to understand the molecular mechanism of PAP function, using a combination of biochemical, biophysical, and genetic approaches, and the Saccharomyces cerevisiae PAP, Pap1, as our model. First, we will use mutagenesis and kinetic analysis to explore the functional importance of motifs in Pap1 which are similar to ones found in RNases, RNA binding proteins, and other polymerases. This approach will be complemented by structural studies using X0ray diffraction analysis of crystals formed from purified Pap1 alone and in complex with ATP and RNA primer. We will also characterize the interactions of Pap1 with subunits of the PF I polyadenylation factor and explore the mechanisms by which these interactions affect Pap1 activity. Finally, we will ask what cellular factors are responsible for the novel ubiquitination of Pap1 in the G2 portion of the cell cycle and determine how this modification affects Pap1 function. Understanding the organization of important domains in a simple polymerase such as PAP can give insights into how functional motifs were combined or modified through evolution to yield the current spectrum of polymerases and nucleic acid modifying enzymes. The structural and mutational analyses will help us understand how PAP selects its substrates, how it differentiates between substrate and product, and how it advances along the single-stranded poly(A) chains as it synthesis progresses. Knowledge of the function and organization of PAP's enzymatic domains is necessary to understand the consequences of modification and protein/protein interactions which modulate PAP function. Information gained about yeast specificity factors will help us to define evolutionary relationships between polyadenylation factors in yeast and higher eukaryotes and to discern why interactions between some factors have changed while others are more highly conserved.

DESCRIPTION (provided by applicant): The emerging model of eukaryotic mRNA synthesis is that transcription and mRNA processing events are carefully orchestrated in vivo by a physical association of the different machineries. For example, RNA polymerase II affects the efficiency of 3' end processing, and processing factors affect the efficiency of transcription termination downstream of poly(A) sites. We are interested in the precise molecular mechanisms involved in the coordination of these two events and have identified several new points of interaction between transcription and cleavage/polyadenylation factors. These findings suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation and facilitate recycling of RNAP II back to the promoter for another round of transcription. This may serve as a mechanism to insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: 1. Can the activity of Ssu72 in transcription be separated from its role in 3" end cleavage? We have found that Ssu72, previously identified as a protein affecting initiation, is directly involved in mRNA 3'end cleavage. We will analyze an existing collection of ssu72 mutants to try to separate the cleavage activity of Ssu72 from a function in transcription initiation and develop new assays to help discriminate these functions. 2. What is the functional significance of the interactions of Sub1 and Ssu72 with Pta1, and how are these interactions regulated? Ssu72 and Sub1 were initially identified based on genetic interactions with TFIIB. We have found that these proteins genetically interact with the Ptal subunit of Cleavage/Polyadenylation Factor (CPF). Moreover, they physically bind Pta1 in a mutually exclusive manner. We will test the hypothesis that sequential interactions of Pta1 with Ssu72 and Sub1 are important for efficient initiation and/or cleavage of pre-mRNA. 3. Does Swd2 function in mRNA synthesis as part of CPF? This protein is intimately associated with CPF and the Set1 histone methylase. However, Swd2 depletion has no effect on 3' end processing, but causes inefficient transcription termination and reduced mRNA levels. We will test the hypothesis that Swd2 affects termination by recruiting Set1 to the transcription complex. We will identify the contact point of Swd2 with CPF and examine how disruption of this interaction affects mRNA synthesis. A genetic screen will be used to identify other important functional interactions with Swd2.

DESCRIPTION (provided by applicant): In this application we propose to create a Short Term Training Institute for undergraduate students in science and engineering to train in several aspects of biomedical research centered around the theme of type 2 diabetes. The goal is to bring engineers and scientists together in multi-disciplinary teams to solve new problems in health-related biomedical research that have not been traditional avenues for bioengineering. The program will be called BEND (Bringing Engineers into New Disciplines), and will involve ten undergraduates for ten weeks during the summer. The students will have didactic and conference courses designed to give them new understanding of biomedical and behavioral problems related to treatment of diabetes. They will also work in small teams with an engineering graduate student mentor and two faculty mentors, one from biomedicine and the other from engineering, to investigate solutions to problems identified by the program faculty through round-table discussions that have occurred during the previous year. The students will present their results at a symposium at the end of the program, and possibly continue to work on the project as a senior honors thesis. Tufts University has two assets that will be combined to create this program. The first is a history of successful undergraduate research training in several individual disciplines, including engineering and biomedicine. The second is a history of and a developing strength in interdisciplinary research. Tufts is poised to create a program that will not only give students in the quantitative sciences direct experience in clinically relevant biomedical research, but also brings together a diverse group of faculty for new avenues of research cooperation.

DESCRIPTION (provided by applicant): The emerging model of eukaryotic mRNA synthesis is that transcription and mRNA processing events are carefully orchestrated in vivo by a physical association of the different machineries. For example, RNA polymerase II affects the efficiency of 3' end processing, and processing factors affect the efficiency of transcription termination downstream of poly(A) sites. We are interested in the precise molecular mechanisms involved in the coordination of these two events and have identified several new points of interaction between transcription and cleavage/polyadenylation factors. These findings suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation and facilitate recycling of RNAP II back to the promoter for another round of transcription. This may serve as a mechanism to insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: 1. Can the activity of Ssu72 in transcription be separated from its role in 3" end cleavage? We have found that Ssu72, previously identified as a protein affecting initiation, is directly involved in mRNA 3'end cleavage. We will analyze an existing collection of ssu72 mutants to try to separate the cleavage activity of Ssu72 from a function in transcription initiation and develop new assays to help discriminate these functions. 2. What is the functional significance of the interactions of Sub1 and Ssu72 with Pta1, and how are these interactions regulated? Ssu72 and Sub1 were initially identified based on genetic interactions with TFIIB. We have found that these proteins genetically interact with the Ptal subunit of Cleavage/Polyadenylation Factor (CPF). Moreover, they physically bind Pta1 in a mutually exclusive manner. We will test the hypothesis that sequential interactions of Pta1 with Ssu72 and Sub1 are important for efficient initiation and/or cleavage of pre-mRNA. 3. Does Swd2 function in mRNA synthesis as part of CPF? This protein is intimately associated with CPF and the Set1 histone methylase. However, Swd2 depletion has no effect on 3' end processing, but causes inefficient transcription termination and reduced mRNA levels. We will test the hypothesis that Swd2 affects termination by recruiting Set1 to the transcription complex. We will identify the contact point of Swd2 with CPF and examine how disruption of this interaction affects mRNA synthesis. A genetic screen will be used to identify other important functional interactions with Swd2.

DESCRIPTION (provided by applicant): Tufts University seeks NIGMS funding for a Summer Internship Program designed to provide biomedical research experiences to Tufts undergraduate students majoring in engineering and computer science. The long-range goal is to encourage cross-disciplinary training for the next generation of biomedical scientists and thus promote an interdisciplinary approach to solving problems related to human health. The specific goal is to increase the number of students who pursue careers in biomedical research. The objectives are: 1. To increase the understanding of students about what a career in biomedical research would entail through distinct, innovative summer research internships on the Tufts Health Sciences Campus; 2. To increase the students' awareness of the benefits of biomedical research and potential careers, collaborations and post-baccalaureate training opportunities through workshops and seminars; 3. To increase students' laboratory skills and confidence by their planning, completing, and presenting a mentored, independent hands-on biomedical research project; 4. To increase the number of students who choose Biomedical Engineering as a major or minor; and 5. To increase the number of graduating engineering seniors who pursue post-baccalaureate training in bioengineering or biomedical research or enter industry careers in these areas. This program consists of a ten-week summer research internship for ten students in which the students interact with and work alongside outstanding research scientists. It builds upon existing strengths at Tufts in engineering, biomedical research, and undergraduate education, a proven commitment of Tufts to undergraduate research as a teaching tool, and a thriving culture of inter-departmental and inter-school collaborations. By exposing the students to potential opportunities and benefits of collaborations with biomedical researchers, it will help the students decide if biomedical research is an area in which they would like to apply their engineering and computer science training.

DESCRIPTION (provided by applicant): The Training in Education and Critical Research Skills (TEACRS) Program serves the national need for university and college faculty trained in biomedical research who are optimally prepared to meet the multiple challenges faced by young Assistant Professors pursuing their first independent position and who understand the value of diversity in the scientific workforce. These individuals must to be fully prepared to meet the demands of setting up and managing a productive research lab, obtaining grant funding, developing and delivering exciting and effective courses and participating in the vibrant life of a institution of higher learning. They need to be able to inspire the next generations of scientists through the teaching and research opportunities they offer. Our goal is to provide talented and qualified postdoctoral scholars with the research portfolio and career skills they will need to succeed in an academic research environment that includes training and mentoring of future biomedical researchers. To achieve this goal, we have partnered with three local minority-serving institutions, the University of Massachusetts, Boston, Pine Manor College and Bunker Hill Community College. Tufts will provide these scholars with rigorous bench research training leading to the development of an independent research program as evidenced by peer-reviewed publications and also provide instruction and activities that build career skills in teaching, written and oral communication, grant writing, ethical conduct of research, laboratory management and mentoring. By working with our partners, we will offer these trainees direct, in-classroom teaching experience with a diverse student body in a mentored setting. TEACRS will also enhance the capacity of our partner institutions to deliver exciting science curriculum and increase accessibility of faculty and students at these institutions to biomedical research. We plan to gradually expand to admitting four trainees each year who will receive receiving 75% support from this grant and 25% support from their research mentor for a maximum of four years.

DESCRIPTION (provided by applicant): In this application we propose to create a Short Term Training Institute for undergraduate students in science and engineering to train in several aspects of biomedical research centered around the theme of type 2 diabetes. The goal is to bring engineers and scientists together in multi-disciplinary teams to solve new problems in health-related biomedical research that have not been traditional avenues for bioengineering. The program will be called BEND (Bringing Engineers into New Disciplines), and will involve ten undergraduates for ten weeks during the summer. The students will have didactic and conference courses designed to give them new understanding of biomedical and behavioral problems related to treatment of diabetes. They will also work in small teams with an engineering graduate student mentor and two faculty mentors, one from biomedicine and the other from engineering, to investigate solutions to problems identified by the program faculty through round-table discussions that have occurred during the previous year. The students will present their results at a symposium at the end of the program, and possibly continue to work on the project as a senior honors thesis. Tufts University has two assets that will be combined to create this program. The first is a history of successful undergraduate research training in several individual disciplines, including engineering and biomedicine. The second is a history of and a developing strength in interdisciplinary research. Tufts is poised to create a program that will not only give students in the quantitative sciences direct experience in clinically relevant biomedical research, but also brings together a diverse group of faculty for new avenues of research cooperation.

[unreadable] DESCRIPTION (provided by applicant): Project summary: Synthesis of mRNA in eukaryotes, and its utilization in the cytoplasm, requires modification at the RNA's 3' end by addition of a poly(A) tail. This process also serves as a point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Even though mRNA 3' end formation occurs in an unexpectedly large complex, most, if not all, of the subunits of this machinery have been identified in the yeast S. cerevisiae. However, little is known about how these 21 proteins cooperate with each other to insure processing that is accurate as well as coupled in a timely fashion to other events in mRNA synthesis and packaging. With the 3'-end processing components in hand, and activities for several of these factors newly defined, a unique opportunity now exists to rigorously address the mechanism by which this essential and universal step in gene expression occurs and is regulated. The central hypothesis of this proposal is that definable rearrangements of protein partners occur within the complex as it evaluates the authenticity of the processing site, commits to cleavage at the poly(A) site, reorganizes to position the poly(A) polymerase for tail synthesis, and releases the final RNA product. The objective is to understand how such reorganizations drive the cycle of mRNA 3' end processing. The research focuses on four events that are likely to be critical transition points in this cycle yet whose underlying mechanisms are not understood. These include the initiation of cleavage and the role of the Ssuy2 protein in this step, the transition from cleavage to poly(A) addition and its regulation by phosphorylation of the Ptai scaffold protein, the control of poly(A) polymerase activity by interactions at its amino-terminus, and the release of processing factors following tail synthesis. The studies will use yeast as the model organism because of the ease of introducing tags and mutations into the genome, the availability of numerous 3' end processing mutants, the ease of purifying processing factors for biochemical studies, and the high degree of conservation of the 3' end processing machinery across eukaryotes. Relevance: Without poly(A) tails, mRNA is targeted for degradation in the nucleus, it does not get out of the nucleus very well, and it is not translated efficiently or turned over at the appropriate rate in the cytoplasm. Maturation of mRNA 3' ends is functionally linked to other essential processes such as transcription, mRNA export, chromosome segregation, DNA repair, and tissue-specific protein expression. Mistakes in polyadenylation can impact on all of these processes. The proposed research should significantly advance our insight into the dynamics of this essential step in mRNA synthesis and identify points at which the constitutive process is likely to be regulated. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. Our long- term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3' ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces. Cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3' end formation could be found. In this study, we will screen the MLSCN Small Molecule Repository for inhibitors of mRNA polyadenylation. This screen utilizes an assay in which defects in 3' end processing in S. cerevisiae lead to production of a reporter required for cell growth. We have adapted this assay to a 384-well format that can be analyzed by an automated plate reader, and it gives robust performance in pilot screens. To assess the spectrum of activity of our hit compounds, we will test them for growth inhibition of mammalian cells and fungal pathogens. We will also use in vivo and in vitro assays for polyadenylation as secondary screens to confirm that hits are indeed targeting mRNA 3' end formation. Finally, we will work with the MLPCN Center to design and synthesize derivatives with increased potency and specificity. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. An added benefit will be the discovery of chemical probes to help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. PUBLIC HEALTH RELEVANCE: Candida has recently emerged as a significant opportunistic pathogen that causes considerable morbidity and mortality in immunocompromised patients. Unfortunately, treatment options for fungal diseases are extremely limited, and compounding this problem, resistance to some of the best anti-fungal drugs is emerging. By taking advantage of certain differences in how fungi and human cells synthesize messenger RNA, we propose to conduct a high throughput screen for a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. An additional benefit will be the discovery of chemical probes that will help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. [unreadable] [unreadable] Our long-term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3' ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3' end formation could be found. [unreadable] [unreadable] In this study, we will develop a high throughput assay to screen S. cerevisiae for small-molecule inhibitors of mRNA polyadenylation. This assay will be based on an existing reporter construct used in our laboratory to detect defects in mRNA 3' end processing. Once the screen is optimized for a 384-well format, we will use it to screen a collection of 140,000 chemicals available through the Broad Institute of Harvard and M.I.T. We will use several in vivo and in vitro assays as secondary screens to confirm that hit molecules are indeed targeting mRNA 3' end formation. We will then determine if the candidate molecules inhibit growth and mRNA 3' end formation in the pathogen C. albicans, and construct a reporter for polyadenylation inhibitors that can be employed in additional large-scale screens directly in Candida. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. [unreadable] [unreadable] Candida has recently emerged as a significant opportunistic pathogen that causes considerable morbidity and mortality in immunocompromised patients. Unfortunately, treatment options for fungal diseases are extremely limited and, compounding this problem, resistance to some of the best anti-fungal drugs is emerging. By taking advantage of certain differences in how fungi and human cells synthesize messenger RNA, we propose to conduct a high throughput screen for a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. [unreadable] [unreadable] [unreadable]

Intellectual Merit. The transcription of eukaryotic mRNA is an obligate step in the flow of information from the genome to expression of proteins needed by the cell. The final step of transcription is called termination, and involves release of RNA polymerase II (Pol II) and the RNA from the DNA template. Defects in termination can impair cell function due to the interference of read-through transcription on downstream DNA elements needed for DNA replication, chromosomal segregation, or the initiation of transcription. Poor termination can also lead to decreased processing and increased degradation of the RNA as well as reduced initiation at the gene's promoter. Despite significant advances in recent years, termination remains one of the least understood steps of transcription. Pol II termination downstream of mRNA poly(A) sites requires the concerted efforts of the Rat1 exonuclease and proteins which recognize and act to cleave RNA at the poly(A) site. Transcription by Pol II through a gene's body is both rapid and processive, yet if stalled, the association of Pol II with DNA is remarkably stable. Nevertheless, interactions of termination factors with the polymerase overcome these challenges and induce changes that lead to pausing and release. The goal of this research is to decipher the molecular mechanisms that lead to Rat1-mediated termination of Pol II and to seek parallels in how Rat1 also facilitates release of Pol I, which transcribes ribosomal RNA. Through genetic screens in yeast, using both directed and random mutagenesis, this project should identify critical regions of RNA polymerase that might interact directly with termination factors or otherwise alter the termination behavior of the enzyme. Previously, the lack of a defined, easily manipulated in vitro system in which to study termination has made it difficult to determine individual contributions of the various factors. This problem has been addressed by developing a new in vitro assay in which mutant and wild-type polymerase can be stalled and then challenged with purified factors alone and in combination. This assay will be incorporated into an integrated strategy that examines transcription in vivo and in cell extract.

Broader impacts. Scientifically, the successful completion of this project should give significant new insight into the mechanism of transcription termination and lead to a new fundamental understanding of points at which this step in the transcription cycle might be regulated in all eukaryotes. Furthermore, it will provide rigorous training for students in the disciplines of biochemistry, molecular biology and genetics as well as multiple opportunities for trainees to gain experience in the written and oral presentation of their research and in mentoring younger students. In addition, the PI has been active in developing new ways to train the next generation of scientists and to increase diversity in biological research, and the members of the PI's lab participate in these programs as mentors and trainees. These programs include summer research and postbaccalaureate programs for underrepresented students, and a postdoctoral training program that prepares fellows for successful academic careers that involve research, mentoring and teaching undergraduates in the biological sciences.

? DESCRIPTION (provided by applicant): Eukaryotic cells often need to modify their gene expression patterns to adapt to environmental changes or to respond to signals regulating cell function. One rapid means of regulating expression is to vary the length of mRNAs. Length at the 3' end is determined by polyadenylation, an essential processing step involving cleavage of mRNA precursor at the polyA (pA) site, followed by addition of adenosines to the new 3' end. Changing the pA site can alter the amount of coding sequence or remove important regulatory sequences in the 3' untranslated region (UTR) that govern localization, translation, and stability. The majority of eukaryotic genes contain two or more pA sites which give mRNA isoforms of different length. Changes in the proportion of these isoforms occurs for a surprisingly large number of genes during development, differentiation, and tumorigenesis and in response to the cell's environment. These findings indicate that alternative polyadenylation (APA) joins transcription initiation and alternative splicing as an important, but under-appreciated way to modulate the amount and types of mRNAs needed for specific cellular states. However, the mechanisms leading to APA are not well defined, and the consequences on protein output and contribution to cell function remain poorly understood. Our overall objective is to understand the mechanism and functional consequences of these widespread changes in pA site usage. Previous work in the field has largely focused on changes to the 3'UTR, and the consequent changes in post-transcriptional regulation due to gain or loss of regulatory sequences. In contrast, relatively little attention has been given to transcripts ending at pA sites within the coding sequence (CDS-pA), despite clear evidence of their prevalence and their systematic variation in response to changes in cell state. We will therefore focus our studies on the CDS-pA transcripts. We will test the hypothesis that variations in the balance of these isoforms are controlled by a coordinated combination of changes in mRNA stability, mRNA polyadenylation, and transcription termination and that regardless of the mechanism, these changes will affect protein production and therefore are an integral component of an appropriate response to environmental changes. Our approach is innovative because it will use whole genome expression analysis to guide focused molecular biological experiments that will probe mechanisms underlying an important step in regulation of gene expression. This proposal is significant because of the wide-spread use of APA and its potential to rapidly affect the amount and type of protein made by a cell. Accomplishment of these aims is expected to yield novel insights broadly applicable to other cellular states modulated by alternative polyadenylation.

A great challenge for a eukaryotic cell is to produce the appropriate amount of each protein and to quickly adjust that production as the cell's environment changes. To meet this challenge, cells must orchestrate many discrete steps, but the details of this integration are not well understood. This research will use baker's yeast as a model to explore the molecular interactions cells use to mount an integrated response to environmental stress. Because all organisms must cope with stresses of various types, the results should be applicable to other systems and may aid in developing strategies for engineering stress tolerance. In addition to advancing scientific knowledge, this project will provide opportunities for training undergraduates, post-baccalaureate trainees, and graduate students from diverse backgrounds. It will improve STEM education by providing research opportunities for junior faculty from primarily undergraduate institutions at critical points in their careers. The project will also provide training in computational science as a quantitative and predictive tool for biological studies, and develop a publicly available web interface for researchers to explore aspects of gene regulation using whole genome datasets.

Proper responses to cellular stress require coordination among a number of gene expression steps. These include different phases of mRNA synthesis--initiation, elongation and termination--as well as multiple steps in processing, such as capping, splicing, polyadenylation, all culminating with export of the mRNA from the nucleus. Each part of this "coordination network" constitutes a step at which the amount and type of mRNA from a particular gene can be regulated. The goal of this project is to use the yeast Saccharomyces cerevisiae as a model organism to identify critical molecular interactions that coordinate transcription and mRNA 3' end processing. In particular, the project will use complementary approaches of molecular biology, biochemistry, genetics, and bioinformatics to determine how core polyadenylation factors contribute to the activation and repression of genes in response to stress. The results should provide new insights into the mechanisms cells use to rapidly fine-tune their responses to changing environmental conditions.

The Tufts IRACDA Program will serve the national need for university and college faculty trained in bio-medical research and optimally prepared for the multiple challenges faced by professors in their first independent position. To be successful, these individuals must set up and manage a productive research lab, obtain grant funding, and develop and deliver innovative science courses. They must understand the value of diversity in the scientific workforce and be able to inspire the next generations of scientists through the teaching and research opportunities they offer. Tufts IRACDA will address these challenges by providing exceptional postdoctoral scholars with the research portfolio and career skills needed to succeed in an academic research environment that includes mentoring of future biomedical researchers. To achieve our goals, we will partner with three local institutions: the University of Massachusetts, Boston, Pine Manor College and Bunker Hill Community College, all of which are committed to educating students from groups underrepresented in the biomedical research workforce. Our Tufts IRACDA program builds upon our successful TEACRS postdoctoral training program. With TEACRS, we developed valuable teaching and research collaborations with our partner faculty and students. Tufts IRACDA will utilize new initiatives to enhance the research and scholarship of our postdoctoral scholars and build upon our existing strengths in teaching and career development. These initiatives will broadly prepare our scholars for academic positions at the nation's top universities and colleges and increase the impact of Tufts connections at our partner schools. To do this, we will: ? Recruit and prepare the nation's best postdoctoral scholars for successful careers in tenure-track, academic positions in the biomedical sciences by providing strong research training. ? Prepare our scholars to create and deliver impactful science curricula to diverse student populations. ? Foster research and mentoring partnerships that encourage undergraduates at our partner institutions to engage in independent research and pursue biomedical science careers. Tufts provides outstanding opportunities for research training in the traditional biomedical disciplines as well as areas such as biomedical engineering, nutrition, and behavioral sciences. Scholars will spend on average 75% of their time conducting research and 25% of their time in career development and teaching activities, including teaching. Similar training, along with comprehensive career mentoring, enabled us through TEACRS to place 88% (36/41) of our alumni in academic faculty positions well-suited to their career goals, a percentage markedly above the national IRACDA average of 73%. In the new Tufts IRACDA program, we will strengthen the training in research and research-related career skills such as grant and manuscript writing, so that we place scholars in a more balanced mix of research-intensive and primarily undergraduate institutions. We plan to support a total of nine scholars each year from IRACDA funds and three from institutional support.

The Tufts IRACDA Program will serve the national need for university and college faculty trained in bio-medical research and optimally prepared for the multiple challenges faced by professors in their first independent position. To be successful, these individuals must set up and manage a productive research lab, obtain grant funding, and develop and deliver innovative science courses. They must understand the value of diversity in the scientific workforce and be able to inspire the next generations of scientists through the teaching and research opportunities they offer. Tufts IRACDA will address these challenges by providing exceptional postdoctoral scholars with the research portfolio and career skills needed to succeed in an academic research environment that includes mentoring of future biomedical researchers. To achieve our goals, we will partner with three local institutions: the University of Massachusetts, Boston, Pine Manor College and Bunker Hill Community College, all of which are committed to educating students from groups underrepresented in the biomedical research workforce. Our Tufts IRACDA program builds upon our successful TEACRS postdoctoral training program. With TEACRS, we developed valuable teaching and research collaborations with our partner faculty and students. Tufts IRACDA will utilize new initiatives to enhance the research and scholarship of our postdoctoral scholars and build upon our existing strengths in teaching and career development. These initiatives will broadly prepare our scholars for academic positions at the nation's top universities and colleges and increase the impact of Tufts connections at our partner schools. To do this, we will: ? Recruit and prepare the nation's best postdoctoral scholars for successful careers in tenure-track, academic positions in the biomedical sciences by providing strong research training. ? Prepare our scholars to create and deliver impactful science curricula to diverse student populations. ? Foster research and mentoring partnerships that encourage undergraduates at our partner institutions to engage in independent research and pursue biomedical science careers. Tufts provides outstanding opportunities for research training in the traditional biomedical disciplines as well as areas such as biomedical engineering, nutrition, and behavioral sciences. Scholars will spend on average 75% of their time conducting research and 25% of their time in career development and teaching activities, including teaching. Similar training, along with comprehensive career mentoring, enabled us through TEACRS to place 88% (36/41) of our alumni in academic faculty positions well-suited to their career goals, a percentage markedly above the national IRACDA average of 73%. In the new Tufts IRACDA program, we will strengthen the training in research and research-related career skills such as grant and manuscript writing, so that we place scholars in a more balanced mix of research-intensive and primarily undergraduate institutions. We plan to support a total of nine scholars each year from IRACDA funds and three from institutional support.

Project Summary: Macrophages are key effector cells of the immune system, with critical functions in killing of microbes, production of inflammatory regulators, and tissue repair. However, an excessive macrophage response contributes to the pathology of cancer as well as inflammatory and degenerative diseases. In addition, unchecked proliferation of macrophage precursors in lieu of differentiation leads to acute myeloid leukemia. To better address how to modulate macrophage function to help abate diseases that involve changes in macrophage biology, we must understand the critical molecular pathways that govern macrophage differentiation and regulate their activity. We propose that one of these pathways will involve mRNA polyadenylation, an essential maturation step in which mRNA precursor is trimmed at its 3' end and a poly(A) tail (pA) added. Changing the position of the pA site through a process called alternative polyadenylation (APA) plays an important, increasingly appreciated role in regulation of gene expression. Shortening of the 3' untranslated region can remove regulatory sequences that control RNA stability, translation, and subcellular localization, whereas coding region shortening can dramatically alter protein function. While global changes in APA have been observed in tumor progression and other types of cellular differentiation, the contribution of APA to macrophage differentiation has not been studied. We hypothesize that a global shift in APA is required for macrophage differentiation and that this shift is driven by changing levels of APA regulators. Our objective is to determine how APA contributes to macrophage differentiation, with the long-range goal of defining how this might be manipulated in therapeutic settings to promote differentiation and modulate macrophage function. Our specific aims will 1) determine the global pattern of APA during macrophage differentiation, the functional classes of genes impacted by APA, and sequence features that might characterize these sites, 2) define drivers of macrophage APA and the consequence that altering their expression has on differentiation as well as well-characterized macrophage functions such as cytokine production, migration, and phagocytosis, and 3) determine the molecular mechanisms that alter the levels of the proteins that regulate APA. Because macrophage are a first line of defense for many diseases and dysregulation of their differentiation leads to leukemias, our proposed studies should ultimately inform new therapeutic tools to modulate macrophage production. They will also broadly advance our understanding of general and tissue-specific APA paradigms. s