Understanding the role of U5 snRNP gene mutation in pre-messenger RNA splicing and craniofacial development

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The DNA of a cell is copied into a pre-messenger RNA (pre-mRNA) that the cell uses as a template for protein production. Some of the information contained in DNA is not required for making proteins, therefore, unwanted information must be removed before a protein is made. This unwanted information is removed, or spliced, from pre-mRNA by a process similar to the editing of unwanted frames from a film. This splicing of the pre-mRNA is very important because it must occur accurately in order for functional proteins to be produced. Splicing at the wrong position could have disastrous effects on the final protein produced. Abnormal proteins generated due to mistakes in splicing could cause defects in the development of an organism or result in disease.The process of splicing is carried out by a large RNA/protein complex called the spliceosome. The spliceosome interacts with the pre-mRNA to identify and splice out the unwanted regions. At the core of the spliceosome is the U5 snRNP. The U5 snRNP contributes to the active site of the spliceosome and orients the pre-mRNA for accurate removal of the unwanted regions from the pre-mRNA which are called introns. Therefore, the U5 snRNP is essential for the function of the spliceosome. We have recently found that mutations in genes that make proteins of the U5 snRNP lead to the craniofacial disorders Burn-McKeown Syndrome (BMKS) and MandibuloFacial Dysostosis, Guion-Almeida type (MFDGA). This observation suggests that, in some situations, mutation in essential splicing factors may only influence a subset of pre-mRNAs. Because patients with these mutations only present with very specific craniofacial defects, it appears that these mutations in the U5 snRNP only influence the splicing of some pre-mRNAs required at a specific developmental stage. It is not clear how only certain pre-mRNAs are influenced by these U5 snRNP gene mutations, thus we propose to investigate this important question during this project. We will take advantage of the high similarity between the human, mouse and yeast U5 snRNP proteins to investigate the exact defects associated with these U5 snRNP gene mutations in the experimentally tractable yeast system and with mouse and human cell lines. To gain an understanding of how U5 snRNP gene mutations cause defects in craniofacial development, we will also explore splicing defects directly in cranial neural crest cells from mice but also develop mouse models of these disorders. We will find pre-mRNAs where the splicing process has occurred at incorrect positions, creating errors that cause the formation of abnormal proteins. We will search for links between these abnormal proteins and craniofacial development to gain an understanding of why craniofacial development is disrupted by the mutations in U5 snRNP genes. Because the process of splicing is critical for cellular survival, gaining a better understanding of spliceosome function through the investigation of mutants with splicing defects informs our understanding of fundamental biological processes. Additionally, this research project will provide essential information on the role of pre-mRNA splicing in development and aid in the understanding of how mutations in core splicing factors can cause disease.