: The broad long term goal of the proposed research is to understand in detail the mechanism of nuclear pre-mRNA splicing in parasitic nematodes using Ascaris as a model system. Cell free extracts prepared from developing embryos of this organism efficiently catalyze both cis and trans-splicing;these extracts remain the only experimental system in which it is possible to analyze trans-splicing using biochemical approaches. Recent studies have identified and characterized two protein factors, SL-30 and SL-95 that are uniquely required for trans-splicing. These proteins function to promote joining of the 5'and 3'splice sites. Recent studies have also revealed that c/s-splicing in nematodes results in the deposition of an exon junction complexon mature mRNAs;to date this complex has only been analyzed in mammalian cells. These observations serve as the basis for future mechanistic analyses, which will first use a depletion/reconstitution approach to study further the function of the trans-splicing specific factors. In addition, fusion protein strategies will be employed to determine if the SL-30 and SL-95 are not only necessary but sufficient for SL RNP function. Second, the evidence indicates that SL-30 makes essential contacts with the splicing factor SF1/BBP, a protein that plays a bridging role between 5'and 3'splice sites in cis-splicing. Depletion/reconstitution will be used to determine which domains of SF1/BBP are required for both cis and trans-splicing. In addition, a sensitive competition assay will be used to determine which region of SF1/BBP communicates with factors at cis 5'splice sites. Finally, the availability of homologous Ascaris extracts that catalyze both splicing and efficient in vitro translation provides a unique opportunity to biochemically address the function of the EJC. Sensitive reporter mRNAs will be used to determine if the EJC enhances translation and, if enhancement is observed, experiments will be conducted to determine the mechanism by which enhancement occurs. In combination, the proposed studies should provide new insight into the mechanism of trans-splicing in particular and splicing in general. They may also provide novel information relevant to the coupling of splicing and post-processing mRNA metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI028799-20
Application #
7559711
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Joy, Deirdre A
Project Start
1990-01-01
Project End
2010-01-31
Budget Start
2009-02-01
Budget End
2010-01-31
Support Year
20
Fiscal Year
2009
Total Cost
$465,086
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Maroney, P A; Romfo, C M; Nilsen, T W (2000) Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol Cell 6:317-28
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Maroney, P A; Denker, J A; Darzynkiewicz, E et al. (1995) Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency. RNA 1:714-23
Denker, J A; Nilsen, T W (1994) Characterization of a DNA-binding factor that recognizes the 22-base pair trans-spliced leader sequence in Ascaris lumbricoides. Mol Biochem Parasitol 66:139-42
Shambaugh, J D; Hannon, G E; Nilsen, T W (1994) The spliceosomal U small nuclear RNAs of Ascaris lumbricoides. Mol Biochem Parasitol 64:349-52
Hannon, G E; Hannon, G J; Maroney, P A et al. (1994) Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence. J Biol Chem 269:12387-90
Nilsen, T W (1993) Trans-splicing of nematode premessenger RNA. Annu Rev Microbiol 47:413-40

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