RNA interference (RNAi) technologies utilize a specific sequence of double-stranded RNA in order to silence the expression of a particular gene. The term RNAi also refers to a broad set of conserved mechanisms that allow cells from most organisms to respond to double-stranded RNA. These mechanisms have vital endogenous roles in cells, including roles that affect chromatin function. For example, RNAi mechanisms help establish heterochromatin, a function that is important in preventing mobilization of transposons. ABC transporters are transmembrane proteins that act as pumps to transport small molecules across a membrane bilayer. Many ABC transporter genes are found in most organisms, and they collectively harbor diverse functions associated with substrate trafficking. Some transporters alleviate cellular stress by exporting toxic substances, whereas other transporters traffic specific substrates that are vital to normal health and development. The substrates trafficked by many transporters, and their precise functions in cells, have yet to be identified. During the course of our analysis of RNAi-defective strains of the nematode worm Caenorhabditis elegans, we observed that some ABC transporter genes are required for an efficient RNAi response to double-stranded RNA as well as for endogenous RNAi activities that affect chromatin function. The current project will enable us to determine how ABC transporters influence RNAi mechanisms. Our work delves into a novel and as yet unexplored area that links two of the most conserved mechanisms in biology.
Broader Impact: Intrinsic to our research goals are projects that will allow for scientific training and career development of undergraduate and graduate students; students will also be engaged in outreach opportunities directed toward enhancing scientific literacy at the K-12 level.
RNA interference (RNAi) describes a remarkable gene silencing methodology that allows scientists to affect the function of virtually any gene by the experimental delivery of sequence-specific double stranded RNA. RNAi also describes those incompletely defined molecular mechanisms in cells and organisms that respond to both experimental delivery of RNAs and to RNAs that are normally produced in cells. RNAi mechanisms normally play vital roles in the cytoplasm and in the nucleus of cells. For example, RNAi mechanisms protect cells against rampant and systemic viral infection in the cell’s cytoplasm. In the nucleus, RNAi mechanisms prevent transposable elements (foreign DNA sequences that have invaded the genomes of most organisms) from activating, excising and reinserting, and potentially mutating the genome. In addition to anti-foreign genome roles, RNAi mechanisms in both the cytoplasm and the nucleus are required for many aspects of normal gene expression. Transposon mobilization and defective gene expression, functions that require RNAi mechanisms, have been associated with genetic defects, a variety of disorders including cancer, and cell death. The studies made possible by NSF funding are relevant to our understanding of how cells guard against the activity of foreign nucleic acid sequences that have invaded our genomes, how RNAi mechanisms affect gene expression, and how RNAi technology might be more effectively utilized to target a gene for silencing. Our studies utilized genetics techniques to analyze RNAi mechanisms in Caenorhabditis elegans, a fast-growing, multicellular animal that is particularly amenable to RNAi. In particular, we investigated RNAi mechanisms with respect to the sub-cellular compartments in which the silencing dsRNAs were generated and the directionality of dsRNA movement into the cytoplasm. Our studies demonstrated that most intrinsically derived dsRNAs that are produced in the nucleus are dependent on cytoplasmic RNAi machinery. We learned that a variety of factors influence the RNAi competency of transgenes, including copy number of dsRNA-expressing cassettes, transgene design, temperature, maternal effects, background mutations and inter-compartmental transport. The work provided educational opportunities for graduate students and undergraduates from Kansas, Missouri, Virginia, Minnesota, New Mexico and other states; their work has been published and they presented their findings in several local, national and international meetings. During the course of our studies, we utilized existing RNAi tools to help us obtain large numbers of C. elegans males. Typically, males are relatively rare in populations of C. elegans, and they arise when an X-chromosome is missing from an egg or sperm. Affecting mechanisms that normally ensure proper chromosome segregation in gamete formation can increase the rate of X-chromosome loss, and RNAi technology is a particularly easy way to knock down the function of genes required for this process in C. elegans. We analyzed existing RNAi reagents and developed new tools to allow for the facile production of males. These reagents will enhance the ability of C. elegans labs to perform phenotypic analysis, genetic crosses and biochemical analyses on males. Undergraduates and three high school students (including two from minority groups under-represented in the sciences) were active participants in these experiments that involved disruption of the same cellular mechanisms that lead to Down, Edward, Patau, Klinefelter, Turner and other syndromes in humans. The organism used in our laboratory, Caenorhabditis elegans, can be engineered to express fluorescent proteins in a variety of different cell types and sub-cellular structures. These brightly colored fluorescent animals have provided a vivid visual starting point for scientific discussions with groups of middle school children from the Kansas City area who tour our laboratory as part of their annual classroom tour of the campus. The NSF-funded projects have also provided laboratory experiences and educational opportunities for a visiting Fulbright scholar from Germany as well as for undergraduate students from Minnesota, Virginia and California who were part of a Research Experience for Undergraduates experience. During the course of our studies, four high school students have participated in scientific experiments in our laboratory—one has since graduated and is currently enrolled in Princeton University. Approximately twenty undergraduates have participated at some level in the scientific enterprise: several have graduated and continued on to graduate programs at the University of Washington, Princeton, Relay Graduate School of Education, Kansas State University, University of Wisconsin, to medical school programs at the University of Iowa and Kansas University; others have participated in programs such as Teach for America, the Jayhawk Health Initiative Brigade; and some have continued with careers in aerospace engineering and materials science. Two students received graduate degrees (MS and PhD) based on their participation in these NSF-funded projects. A new crew of high school students, undergraduate students, and graduate students continue to avidly pursue related scientific questions.
