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Investigating the Link Between FKBPs and Splicing in Bombyx Mori: Implications for Agricultural Pest Control

EPA Grant Number: F6D10905
Title: Investigating the Link Between FKBPs and Splicing in Bombyx Mori: Implications for Agricultural Pest Control
Investigators: Somarelli, Jason
Institution: Florida International University
EPA Project Officer: Boddie, Georgette
Project Period: September 1, 2006 through September 1, 2009
Project Amount: $95,983
RFA: STAR Graduate Fellowships (2006)
Research Category: Fellowship - Insect Science , Fellowship - Molecular Biology , Academic Fellowships , Biology/Life Sciences

Description:

Objective:

Our main project goal is to characterize the function of the immunosuppressive drug binding protein FKBP45 in pre-mRNA splicing. The insect order Lepidoptera includes members that are among the most destructive agricultural pests in the world (Mita et al. 2003). The United Nations estimates that approximately 40% of agricultural production is lost to insect pests, pathogens and weeds each year (Day et al. 1997). With the global population on the rise, the need for higher agricultural productivity is a top priority. Toxic pesticides are often employed as a means with which to increase agricultural productivity, but this strategy only adds to greater long-term environmental damage such as the development of pesticide resistant insect populations and pollution of drinking water. Increased knowledge of pre-mRNA splicing in Lepidopterans may allow researchers to silence sex-determinant alternatively spliced transcripts and ultimately create single-sex pest populations.

Several insect systems utilize alternative splicing as a means with which to determine the sex of the developing embryo. In Bombyx mori, the Bmdsx gene is a homologue of the sex determining dsx gene in Drosophila which is involved in somatic sex differentiation through a complex and intricate alternative splicing pathway (Funaguma et al. 2005; Saccone et al. 2002). Our laboratory has identified a protein that differentially binds to U1 snRNA and possesses overlapping RNA and immunosuppressive drug binding domains (Somarelli et al. submitted to Insect Molecular Biology). Additionally, Graveley (2005) recently noted that a nontoxic drug derivative of rapamycin can be utilized to control pre-mRNA splicing in vitro. We also intend to create an outreach program to educate students about our research and encourage them to pursue careers in the environmental sciences.

This work seeks to further understand Lepidopteran transcription regulation in an effort to possibly silence sex-determinant alternative splicing and possibly create single sex insect populations. In addition, understanding Lepidopteran biology can be useful in comparative insect research as well as biodiversity and ecology studies.

Approach:

1. To characterize tissue- and developmental stage-specific expression of FKBPs in Bombyx mori.
2. To identify FKBPs in splicing complexes using native polyacrylamide gels.
3. To determine whether the immune suppressant rapamycin binds FKBP45 from Bombyx mori.
4. To assess whether FKBP45 is essential for pre-mRNA splicing using in vitro splicing assays.

Expected Results:

Preliminary data using Western blotting suggests that FKBPs exhibit spatio-temporal differential expression. In addition, initial experiments reveal FKBPs exclusively in nuclear extract fractions containing high concentrations of spliceosomal complexes obtained by glycerol gradient ultracentrifugation. Given the high sequence similarity between FKBP45 and FKBP46 from Spodoptera frugiperda within the FKBP drug binding domain, we fully expect that rapamycin will bind FKBP45, in a manner similar to FKBP46. Since FKBPs have been identified in fractions containing high molecular weight spliceosomal complexes, it is our anticipation that FKBPs are implicated in splicing; however, it is also possible that FKBPs are associated with the spliceosome as protein trafficking molecules or for some other function.

Supplemental Keywords:

FKBPs, immunophilins, pre-mRNA splicing, Bombyx mori, Lepidopteran, , Ecosystem Protection/Environmental Exposure & Risk, TREATMENT/CONTROL, Sustainable Industry/Business, Scientific Discipline, RFA, Technology for Sustainable Environment, Sustainable Environment, Biology, Technology, Monitoring/Modeling, Genetics, biotechnology, drinking water, genetic engineering, agriculture, environmental technology, alternative materials

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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