Mark B. Roth - US grants

The work described here is designed to further our understanding of the structure function assembly, and intracellular movements of proteins that associate with nascent polymerase II transcripts. These proteins play a critical role in the expression of genetic formation. I will study tow different types of proteins; those that interact with most polymerase II transcription units, and those that associate with only one or a few. The specific approaches are: 1) I will express two proteins that interact with most nascent polymerase II transcripts in amphibian oocytes and follow their progression over time as they move to the nucleus, associate with other transcript binding proteins, and interact with the nascent transcripts. To distinguish them from the endogenous forms of these proteins I will use constructs that express thee proteins with an additional 10 amino acids that can be recognized by antibody that does not cross react with any endogenous proteins. 2) I will further characterize a protein that interacts with a specific set of transcription units on chromosome 14 of Xenopus laevis. This phosphoprotein is present widely in the animal kingdom, and is associated with a large novel nuclear substructure.

The work described here is designed to further our understanding of the structure function assembly, and intracellular movements of proteins that associate with nascent polymerase II transcripts. These proteins play a critical role in the expression of genetic formation. I will study tow different types of proteins; those that interact with most polymerase II transcription units, and those that associate with only one or a few. The specific approaches are: 1) I will express two proteins that interact with most nascent polymerase II transcripts in amphibian oocytes and follow their progression over time as they move to the nucleus, associate with other transcript binding proteins, and interact with the nascent transcripts. To distinguish them from the endogenous forms of these proteins I will use constructs that express thee proteins with an additional 10 amino acids that can be recognized by antibody that does not cross react with any endogenous proteins. 2) I will further characterize a protein that interacts with a specific set of transcription units on chromosome 14 of Xenopus laevis. This phosphoprotein is present widely in the animal kingdom, and is associated with a large novel nuclear substructure.

This a letter of application to the MR to conduct immunoelectron microscopy on early C elegans embryos with a monoclonal antibody we developed that binds the C elegans centromere. This is a collaboration with John White. The purpose of the project is to determine where the antigen recognized by this antibody is concentrated relative to the Idnetochore. Our previous fight microscopic analysis of antibody stained embryos suggested mitosis specific expression of the antigen with the highest concentration on the poleward face of the holocentric chromosomes at metaphase. A limitation of fight microscopy is that we cannot distinguish whether the antigen is concentrated in the kinetochore or somewhere near it. DVM researchers experienced in immunoelectron microscopy may be able to overcome this limitation.

The work described here is designed to understand the mechanisms of pre- mRNA splicing. This is important for human health in so far as changes in splicing and RNA processing in general have been correlated with disease. Our goal is to identify and characterize the factors involved in pre-mRNA splicing with the hope that this information will be useful in the development of therapy options. We outline biochemical, cell biological, and genetic approaches designed to research this goal. To better understand the mechanisms we intend to continue to test the hypothesis that SR proteins alone can function to join exons together prior to splicing. For this we are using biochemical assays and electron microscopic analysis of SR protein bound pre-mRNA complexes. Previous studies show that disease can be associated with changes in nuclear structure. For this reason we intend to study the localization of splicing factors to active sites of gene transcription. Finally, determination of how cells regulate pre-mRNA splicing will be incomplete until all required pre-mRNA splicing factors are identified. Only a few splicing factors have been identified to date, and splicing is still carried out in crude nuclear extract. We isolated an antibody that has made it possible to isolate genes encoding more than half of the proteins present in purified splicing complexes. We intend to analyze structure and pre-mRNA splicing function of these proteins using standard biochemical approaches as well as using a gene inactivation approach in C. elegans.

DESCRIPTION (provided by applicant): Equal segregation of chromosomes to opposite poles during mitosis is essential for cell viability. To accomplish this chromosomes are condensed and attached to spindle microtubules at the centromere which coordinates the separation of sister chromatids at anaphase. Missegregation of chromosomes has been associated with many human diseases including several congenital disorders and nearly all forms of cancer. To better understand how chromosome segregation occurs we have begun to study the centromere in the freeliving soil nematode C. elegans. The centromere in C. elegans is approximately 10 times larger than it is in human cells making this system more amenable to cell biological studies. In addition functional studies can be carried out rapidly because it has recently become possible to eliminate gene functions using RNA mediated interference. My goal is to identify the proteins and DNA sequences involved in the assembly of the centromere and to identify the mechanisms involved in forming new centromeres. The approaches I will use are a combination of biochemical and cytological studies using RNAi and temperature sensitive mutants affected in these processes.

DESCRIPTION (provided by applicant): Dietary restriction can slow reproductive development, extend longevity, and improve the outcome of metabolic and neurological disease. Upon severe dietary restriction, many organisms, including mammals, initiate programs of developmental or reproductive quiescence, or diapause. In these arrested states, animals withstand prolonged starvation, and recover without significant detriment to reproductive output or adult longevity. In preliminary studies, we have found that the C. elegans HNF41 ortholog, NHR-49, is essential for longevity and reproductive protection in the adult reproductive diapause, an intriguing physiological state that has yet to be described in C. elegans. There are a number of features associated with this adult diapause that make it of broad interest, most notably, its impact on reproductive activity, embryonic development, stem cell maintenance, and energy metabolism. Thus, investigation of NHR-49 in C. elegans provides a unique opportunity to define the involvement of nuclear receptors in nutritional diapause. Specifically we will address three aspects of this NHR-49 dependent adult arrest. In Aim 1, we will conduct experiments to understand how germline stem cells are protected during extended periods of starvation, distinguishing between models of germ cell quiescence and self-renewal, and assessing the role of the stem cell niche. In Aim 2, we will test our hypothesis that an NHR-49 dependent stimulation of fat expenditure is essential for starvation dependent extension of lifespan and reproductive longevity. Additionally, we will test the intriguing hypothesis that constitutive induction of fat expenditure may be sufficient to increase longevity, even in the absence of dietary restriction or starvation. PUBLIC HEALTH RELEVANCE: Caloric restriction and intermittent fasting can extend longevity and improve the outcome of metabolic and neurological disease. We have discovered a gene that is important for preserving cellular function and youth during periods of dietary restriction. We propose that elucidating the function of this gene may enable the development of pharmacological treatments or dietary interventions that can mimic the positive effects of dietary restriction.