Contribution of splicing factors to developmental defects: links between the spliceosome protein Prpf8 and cilia formation

The yeast protein Prp8 (named Prpf8 in mouse and PRPF8 in human) is the master regulator of the spliceosome. Located at the heart of the spliceosome, Prp8 is an essential, highly conserved, 280 kDa protein that interacts directly with the 5’ splice site, 3’ splice site and branch site in the pre-mRNA. Recent structural studies have verified the critical role of Prpf8 in the active spliceosome. In addition to its role in splicing, new data has shown that Prpf8 is localised within the cytoplasm to the base of the cilium, and that a knockdown of Prpf8 function leads to defects in cilia formation.

We have discovered a mouse mutant with defects in cardiac left-right development. Surprisingly we found that 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 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. Given the recent identification of Prpf8 as a protein localised to cilia, and the known function of cilia in left-right axis formation, we propose that transcripts encoding cilia proteins will be among those that are misspliced in Prpf8 mutants. 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.

This project will require the student to apply computational and mathematical techniques to high-quality quantitative biological data. The student will generate experimental RNA-seq datasets, and will then learn to analyse these datasets using multiple computational analysis tools to identify splicing and expression defects. Functional enrichment analyses will be performed to identify the biological processes and pathways that are altered in mice with mutations in the spliceosome protein Prpf8.

The student will also learn techniques required for the investigation of gene function during development, such as immunofluorescence and in situ hybridisation. The project will also provide training in biochemical methods such as co-immunoprecipitation and Western blotting. Training in standard laboratory protocols and general molecular biology methods will also be provided.

Overall this project will incorporate multiple techniques using different experimental systems to provide a diverse and broad skill set to the student.

Candidates are expected to hold (or be about to obtain) a minimum upper second class honours degree in biology, genetics, or a related subject area. Candidates with prior laboratory experience and an interest in genetics, computational biology, biochemistry, or developmental biology are encouraged to apply.