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

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

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.

Technical Summary

Pre-mRNA splicing is essential for gene expression during development, differentiation, responses to the environment and aging. Mis-regulation of splicing is associated with numerous diseases. Splicing is catalysed by the spliceosome that assembles with a pre-mRNA, identifies the splice sites, then arranges into specific conformations for intron removal. The U5 snRNP is at the heart of the spliceosome and contributes to the active site. We have recently found that mutation which reduces expression of the DIB1 gene, that codes for a U5 snRNP protein, causes the craniofacial disorder BMKS. Mutation that reduces expression in another U5 snRNP protein gene SNU114 is also known to cause the craniofacial disorder MFDGA, pointing now to a common pathway involving pre-mRNA splicing that influences craniofacial development. We propose that mutation in DIB1 and SNU114 influence the splicing of a subset of pre-mRNAs that are required for craniofacial development. However, the identity of these pre-mRNAs and the mechanism by which reduced expression of DIB1 and SNU114 influences their splicing is not known. To discover the defects in splicing, the specific pre-mRNAs that are misspliced and the influence of reduced DIB1 and SNU114 expression on craniofacial development we will:

1) use in vitro and in vivo approaches in yeast, human and mouse systems to uncover spliceosome defects caused by reduced DIB1 and SNU114 expression

2) use RNA-Seq and RT-PCR in the yeast and mouse systems to find the genes that are misspliced when DIB1 and SNU114 expression is reduced

3) use in situ hybridisation and immunohistochemistry/fluorescence to define expression patterns of DIB1 and SNU114 mRNAs and proteins during mouse craniofacial development

4) develop mouse models for BMKS and MFDGA

Overall this work will provide information on how mutations in essential splicing factor genes influence the splicing of genes required for craniofacial development and cause disease.

Planned Impact

Who will benefit from this research?

We have identified numerous groups of users and beneficiaries outside the academic research community who will benefit from the research in this proposal. These are primary school children, secondary school children, University students, clinical researchers, industrial collaborators and third-sector organisations.

How will they benefit from this research?

The research in this proposal is basic research. The knowledge obtained through this research will provide the fundamental theories and concepts underlying cell function, gene expression, development and disease. We can impart this new knowledge to our student beneficiaries through the numerous engagement activities we undertake (see Pathways to Impact). In addition, the fundamental theories and concepts we discover will provide information for more disease-oriented investigations by clinical researchers. Our research into the regulation of RNA splicing may also benefit commercial private sector researchers who are current collaborators. Third sector organisations such as Genetic Alliance UK, Genetic Disorders UK, Rare Disease UK, Sparks and Newlife Foundation who are interested in causes/treatments of rare genetic diseases, will also be interested in the results of our research.

What will be done to ensure that they have the opportunity to benefit from this research?

Our lab has engaged with primary school children through presentations about DNA and genetics at a local school. We have engaged with secondary school children through the "Researchers in Residence" programme, through presentations at Manchester Museum, through workshops at NOWGEN Centre for Genetics in Healthcare and through writing articles for the "Biological Sciences Review". We also engage with secondary school children from deprived areas of Manchester through an annual programme where students perform a developmental biology practical at the University. We have engaged University students by discussing and presenting our work through practical and lecture courses at our University. All these engagement activities will continue and develop through feedback from the beneficiaries. We will directly contact clinical researchers, third sector organisations, our current industrial collaborators and newly identified industrial links to inform them of our research. We will work closely with the University of Manchester Intellectual Property (UMIP) to investigate commercial options resulting from research projects and negotiating intellectual property rights.

Publications

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Description The related craniofacial disorders Burn-McKeown Syndrome (BMKS) and MandibuloFacial Dysostosis Guion-Almeida type (MFDGA) are characterized by mandibular and malar hypoplasia, microcephaly, choanal atresia, external ear anomalies and other variable craniofacial and developmental defects. We have recently found that mutations that reduce expression of the U5 snRNP gene TNXL4A (DIB1) cause BMKS1. Interestingly, reduced expression of the U5 snRNP gene EFTUD2 (SNU114) causes MFDGA providing a strong link between U5 snRNP function and these craniofacial disorders, as well as a possible functional link between the Dib1 and Snu114 proteins during splicing. The mechanisms by which reduced expression of essential splicing factor genes, required for splicing all pre-mRNAs, bring about these particular craniofacial disorders are unclear. Our hypothesis is that reduced expression of DIB1 and SNU114 partially disrupts spliceosome function, resulting in missplicing of a subset of pre-mRNAs required during craniofacial development.

Reduced expression of DIB1 and SNU114 in yeast does indeed cause missplicing of some pre-mRNAs but not others. In addition, we have found that reduced expression of DIB1 and SNU114 cause defects in snRNP assembly, including defects in tri-snRNP formation. We are currently investigating whether there is a functional link between Dib1 and Snu114 within the spliceosome. Growth of yeast models under a variety of conditions has identified conditions that induce ER stress as affecting growth of yeast models more than wild type cells. Related to ER stress sensitivity we have identified the intron containing gene, CNB1, as being particularly susceptible to reduced DIB1 and SNU114 expression. CNB1 codes for Calcineurin B, the regulatory subunit of calcineurin, a Ca++/calmodulin-regulated type 2B protein phosphatase. Calcineurin is important in the signaling pathway that promotes cell survival under stress.

Further, we employed RNA-Seq in yeast models of BMKS and MFDGA to identify global changes in splicing following temporal knockdown of either DIB1 or SNU114. RNA-Seq confirmed extensive missplicing of subsets of pre-mRNAs in both yeast models and importantly, defective splicing of CNB1 which inversely correlates with relative levels of DIB1 or SNU114.

Reduced levels of Calcineurin cause ER stress, which in turn induces apoptosis. Apoptosis of neural crest cells, at certain times and locations during development, is critically important for proper craniofacial development. We are now investigating whether patient cells are more sensitive to ER stress and we are setting up mouse models to determine exactly how reduced DIB1 and SNU114 expression leads to BMKS and MFDGA.
Exploitation Route Clinicians could use this information to develop treatments and improve diagnosis of craniofacial disorders.
Sectors Healthcare

 
Description Enhancing quality of life and health. Using yeast models we have determined that genetic missense mutations associated with the craniofacial disorder MandibuloFacial Dysostosis, Guion-Almeida type (MFDGA) are not deleterious to Snu114 protein function as previously thought. This work provides important information for the future characterisation of missense mutations in patients with this genetic disorder.
First Year Of Impact 2016
Sector Healthcare
Impact Types Societal