Each of us was once a single cell no bigger than the period at the end of this sentence; it is incredible that we have developed into adults with all the correct tissues and organs. To understand the development of the germline, an essential tissue for the continuity of the species, the Bennett laboratory studies P granules, germline-specific RNA and protein aggregates in the soil nematode (round worm) Caenorhabditis elegans. The Bennett group discovered GLH (germline RNA helicase) proteins are P granule components; recently they found GLH-1 interacts with Dicer (DCR-1) in C. elegans; this interaction was discovered both by genetics, with mutants missing either protein, and by biochemistry. Dicer is an enzyme that processes and activates microRNAs, small RNAs recently shown necessary for normal plant and animal development. This project consists of three Aims: to determine 1. how GLH-1 and DCR-1 are interdependent, 2. what RNAs the GLH/DCR complex binds and 3. how GLH-1/4 contribute to the identity of the C. elegans gonad, which can change from a single to a bifurcated tube when the glh-4 gene is missing, with glh-1 loss by RNA interference, or with loss of two microRNAs predicted to bind and regulate glh-1. Therefore, the laboratory will investigate this change in tissue morphology associated with GLH-1/GLH-4 loss. Success in these Aims may provide a molecular understanding of how the germline is specified and how tissues take form. This project will support the education of three MU PhD students; all three are women. It also funds a collaborative project with Central Michigan University, a primarily undergraduate institution. Dr. Bennett trains young scientists in her laboratory and throughout the US, including teaching C. elegans genetics to high school AP Biology students and organizing two-day Young Faculty Boot Camps that precede the Society for Developmental Biology national meetings.
Intellectual Merit: The Bennett group studies P granules, which are germline-specific RNA and protein containing aggregates that hug the outside membranes of the nuclei of cells in the germline and necessary for the production of normal oocytes. Without the GLH proteins, which are P-granule components in the well studied small roundworm C. elegans, or the corresponding, very similar proteins in all other animals, there is no next generation; the animals are sterile. The Bennett group found that the GLH-1 protein binds other proteins, including the well-known protein Dicer, a protein that is important in the production of microRNAs (miRNAs). MicroRNAs are small RNAs that do not code for proteins. They have been rather recently discovered and are now known to be important in the development of both plants and animals; some miRNAs have critical roles in cancer and other diseases. GLH-1 and Dicer completely depend on each other in the C. elegans germline, the tissue that produces all the eggs and sperm. If GLH-1 is missing, so is Dicer; the reverse is also the case. This NSF funding will result in four publications, three of which are already published, or accepted for publication, with the final manuscript in preparation. One of the publications resulting from this NSF award, Beshore et al., 2011, describes the relationship between GLH -1 and Dicer considered above. Another publication, which was just recently accepted, Gao et al. found another new P- granule component PAN-1, which is also a GLH-1 binding partner and causes sterility when the protein is missing. The results described as in preparation have used the new high-throughput, sequencing method called RNA-seq to analyze all the microRNAs (miRNAs) in the C. elegans germline. Surprisingly, there are only a few (less than 10 different miRNAs out of 150 miRNA families in C. elgans) microRNAs that are exclusive to the germline. We currently are verifying this specificity and looking at the effects of loss of these miRNAs before publishing this finding. Broad impacts: The broad impacts of this project include the generation of new anti-DCR-1 and anti-PAN-1 antibodies that have allowed the visualization of the Dicer and PAN-1 proteins in the tissues of C. elegans. Before this work, there were no anti-PAN-1 antibodies and those for the C. elegans Dicer protein did not work in the worm itself, but only for western blot analysis, which analyzes denatured proteins, in our case taken from the worm. Both these antibodies have been made available to those researchers who have requested them. Throughout her career and specifically during this granting period Dr. Karen Bennett has served as an advocate for public outreach as a means to communication about the importance of scientific research and for broadening diversity of the participants in the scientific workforce. She has served on the executive committee of the PREP Scholars program at the University of Missouri (MU), a training program for underserved minority students, since its inception (about ten years ago). She also serves on the newly-established MU School of Medicine Diversity committee. In the last three years she has carried out outreach in OH, NY, UT, and IN to elementary school children in the 4th grade and in 2010 even took a "worm show" with microscopes, a TV monitor and various visibly mutant worm strains to the NSF Child Development Center in Arlington VA, showing nematodes to the young children and introducing our work with worms to 5 classes of preschoolers, from the age of 2-6. She regularly spends several days a year with AP Biology high school classes to set up and analyze genetic crosses using C. elegans. In addition, many of the undergraduates in her laboratory are included as authors on the papers that result from the research carried on in the Bennett group; during this funding period three undergraduates have been contributing authors, one of whom is a female and another an African American male.
