The process of messenger RNA splicing, whether general or alternative, regulates gene expression in a highly conserved manner from flies to humans and maximizes the number of proteins that one gene can produce. Yet little is known about the earliest and most critical steps of splicing regulation, when intron and exon borders of pre-mRNA are first recognized and defined by the spliceosome. Although over 200 proteins associated with the spliceosome have been identified, exactly how these proteins function with one another in a living organism remains unclear. The testis of the fruit fly Drosophila melanogaster provides an ideal in vivo model system to examine splicing regulation in a tissue specific manner. This project takes advantage of the wide spectrum of genetic manipulations and biochemical analysis possible in flies to examine the function of proteins integral to tissue specific splicing. The overall aims are: 1) to detect alternative splicing events in these fly testis tissue, 2) to identify transcripts that are responsive to reductions in the level of early splicing factors and 3) to identify regulatory splicing proteins associated with the early spliceosome.
The experiments listed above will be conducted primarily by undergraduate science majors during summer/academic year research experiences outside of normal coursework at Widener University. The University provides free summer housing to students engaged in summer research and will grant release time from the principal investigator's normal teaching load to supervise research students. In the past five years at Widener, a significant number of student researchers have been women, underrepresented minorities or first generation college students. All of these demographics are represented in the students in this new principal investigator's first research group. In addition to providing supplies, equipment and salaries, this RUI project will expose underrepresented groups to the research process and allow them to attend and present their research at meetings such as the Annual Drosophila Research Conference. The project will provide support for undergraduate students to actively participate in an important research problem and their results will greatly enhance the understanding of splicing within the framework of a living organism.
In an essential process conserved from flies to humans, splicing turns genes off and on through a complex and dynamic mechanism involving RNA (the intermediate between DNA and proteins) and around 200 different proteins. Over 90% of human genes are alternatively spliced, allowing one gene to produce many different products to maximize the output of a genome. Alternative splicing occurs in different tissues, sexes and stages of development and mutations that affect splicing cause many human diseases including cancer, neurodegeneration and obesity. Despite the critical nature of splicing, much remains to be understood about how splicing is regulated or changed in specific tissues in a living organism, particularly in response to external signals like nutrient availability. Several recent discoveries have shown that changes in the level of splicing proteins are associated with obesity in humans and can lead to an increase or decrease in fat storage in the fruit fly model organism Drosophila melanogaster. Drosophila is a powerful model organism for the understanding of essential processes like splicing because government funding of fruit fly research has provided a wealth of publically available resources such as a well-characterized sequenced genome and fly lines that are easy to manipulate genetically and biochemically in the lab. During the course of this award, over twenty undergraduate students in my research lab have gained valuable research and scientific analysis skills while examining splicing and nutrient availability in Drosophila at Widener University, a teaching-centered primarily undergraduate institution. We focus our studies on the fat body, a tissue that controls fat storage and energy metabolism in insects and is similar to the human liver and adipose (fat) tissue. We have developed the fat body as an optimal system in which to examine splicing regulation in a tissue specific manner. We have shown that mutations in splicing proteins result in altered fat storage during the development of the fly and that this effect may be sex-specific. By searching the publically available bioinformatic database FlyBase, we have generated a list of over 300 candidate metabolic genes that are alternatively spliced in the fat body and may contribute to the observed changes in fat levels. We have characterized the splicing pattern of the gene CPT1 and correlated the decreased level of a splicing protein with a change in CPT1 splicing pattern and increased fat storage. This work demonstrated that the fat body of Drosophila is an optimal but largely unexplored tissue for investigating alternative splicing and has enhanced the understanding of splicing critical for proper fat storage within the framework of a living organism. Undergraduate students trained with this project have gained research lab skills in fruit fly genetics, molecular biology, biochemical assays and bioinformatics beyond their coursework. This award supplied materials for the research projects of twenty-three students and summer salary for eleven students, eight of whom are first generation college students and four of whom are underrepresented minorities. Students gained practical experience and mentoring in scientific research rather than working unrelated summer jobs. This increases their confidence in their scientific abilities and frequently results in them becoming leaders in their science lecture and lab courses. Students who work in the lab during the summer often continue their project during the academic year as an independent study or senior thesis. This award also supported travel to the national Drosophila Research Conference and other professional meetings where students gained valuable experience presenting their research to experts in the field and benefitted from undergraduate specific programming. These in-depth experiences lead to better post-graduation outcomes and all fifteen of the student researchers who graduated in 2010-2014 have been employed in science or attended a science related graduate program or medical school within one year of graduation. This award enhanced the learning environment at Widener by supporting students both in the research lab and classroom. Although alternative splicing is not covered in depth in the undergraduate curriculum, students working on this project read the primary literature and conducted experiments to better understand the field. Their peers, both science and non-science majors, also are exposed to information about splicing and the use of Drosophila as a model organism at a higher level through presentations by student researchers on-campus. My involvement with the project during the summer lead to me co-developing the Summer Research Program (SRP) for all students in the Arts and Sciences at Widener. The SRP prepares students for post-Widener life with informational sessions on topics such as applying to graduate school, interviewing for jobs and writing a resume. Alumni are invited back to speak to students about life in graduate school or working in a lab. As a result, more students are applying to graduate school and external summer research programs. Therefore, this award has contributed greatly to the development of the next generation of scientists.
