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The role of the spliceosomal protein Prpf8 in mammalian development


Project Description

We have discovered a mouse mutant with defects in cardiac development, including reversal of the left-right axis. This mutant has a missense mutation in the spliceosomal protein Prpf8, which we predict causes aberrant splicing in mutant embryos. Modelling this mutation in yeast confirms that the mutant protein shows reduced splicing efficiency in a splicing reporter assay. Based on the recently solved structure of yeast Prpf8, we propose that our mutation alters the interaction of Prpf8 with another splicing factor. In this project we will test this hypothesis biochemically by examining physical interactions between wild type and mutant forms of Prpf8 and other spliceosome proteins. We will also use the sensitive yeast reporter assay to characterise which steps of the splicing process are altered by the Prpf8 mutation. Regions of the Prpf8 protein that are highly evolutionarily conserved will also be mutated and then examined using the yeast splicing reporter assay to determine if they disrupt Prpf8 function. We will also identify transcripts that show aberrant splicing in the Prpf8 mouse mutant, and determine their expression pattern. We will investigate the function of aberrantly spliced genes to identify links to cardiac development and left-right axis formation. Further characterisation of the Prpf8 mutant mouse to define the stage at which the left-right axis is altered during development will also form part of this project. Overall, these experiments will lead to new insights into the biochemical function of Prpf8 during splicing, and the requirements for splicing fidelity during cardiac development and left-right axis formation.

Funding Notes

This project has a Band 3 fee. Details of our different fee bands can be found on our website. For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website. Informal enquiries may be made directly to the primary supervisor.

References

Grainger RJ, Beggs JD. Prp8 protein: at the heart of the spliceosome. RNA. 2005 May;11(5):533-57.

Kershaw CJ, Barrass JD, Beggs JD, O'Keefe RT.Mutations in the U5 snRNA result in altered splicing of subsets of pre-mRNAs and reduced stability of Prp8. RNA. 2009 Jul;15(7):1292-304.

Kile BT, Hentges KE, Clark AT, Nakamura H, Salinger AP, Liu B, Box N, Stockton DW, Johnson RL, Behringer RR, Bradley A, Justice MJ. Functional genetic analysis of mouse chromosome 11. Nature. 2003 Sep 4;425(6953):81-6.

Sutherland MJ, Ware SM. Disorders of left-right asymmetry: heterotaxy and situs inversus. Am J Med Genet C Semin Med Genet. 2009 Nov 15;151C(4):307-17.

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