Analysis on how RNA splicing factors change global gene expression patterns and regulate male fertility.

Infertility affects many couples, but the reasons causing it are usually unknown, as are the mechanisms required for normal fertility. This project will analyse RNA binding proteins implicated in male infertility, to understand their role in normal spermatogenesis. We will take advantage of existing mouse models and make cell lines that we can manipulate in vitro. This project will include world class training in both molecular biology and bioinformatics which will place the student in a strong place in the jobs market.

Background: Recently the Elliott lab found that an RNA binding protein called RBMXL2 is essential for normal spermatogenesis, but why is not fully clear (PMID: 34927545). In the Veltman lab, heterozygous de novo mutations affecting RNA binding proteins have been identified that prevent normal healthy development of the testis (for example, mis-sense mutations in the RBM5 gene, PMID: 35013161). What gene expression pathways are affected by these RNA binding proteins, and why they affect normal healthy development of sperm is unknown. Both RBMXL2 and RBM54 are involved in controlling splicing. Splicing is critical since most human genes are split between exons and introns. A macromolecular machine called the spliceosome recognises exon sequences and splices them into mRNAs. Most genes can be spliced in more than one way, and changes in splicing patterns can alter gene function. Splicing factor proteins control whether exons are recognised or not, and this can change gene function in important ways. But these RNA binding proteins could also have additional unexpected roles in normal healthy germ cells.
Hypothesis and aims: We hypothesise that mutations occurring in the human RBM5 gene and the mouse RBMXL2 gene will change impact normal healthy development of the testis. Our aim is to use mouse tissue and human cell lines to investigate this.
Methodology: The student will analyse patterns of gene expression and cell biology in RBMXL2 knockout mice and wild type mice to identify defects caused by loss of RBMXL2 within an animal model. These will include splicing and gene expression changes, and corresponding changes in cellular processes important for spermatogenesis. The student will use genome engineering of human cultured cells to investigate the function of RBM5 and how this is changed by dominant negative mutations. A pool of predicted differentially spliced genes will be validated using RT-PCR and capillary gel electrophoresis. The student will interrogate mechanisms how normal patterns of gene expression of key genes is achieved, using minigenes made by genomic amplification and transfected into cells with the wild type and mutated versions of RBM5 and RBMXL2. As a backup to analysing endogenous targets the student will also characterise the effects of de novo mutations on RBM5 and RBMXL2 function on candidate minigenes that are already available.
Potential impact: Up to 7% of men suffer from infertility. Despite this, the mechanisms of male infertility are very poorly understood. This project will follow on from recent work in our collaborating groups to start to address what global pathways of gene expression pathways are mis-regulated as a result of these de novo mutations. If successful, we can expand on this work by performing similar studies in other splicing genes found to be mutated in male infertility. This work will contribute to improving the genetic diagnosis in infertile men, which will provide more insight into the potential success of assisted reproductive technologies, the chance of passing on infertility with these approaches and co-morbidities associated with the infertility.