A transgenic approach to investigate the RNA binding protein T-STAR

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Human genes are found on chromosomes, and encoded by DNA. Recently the almost complete DNA sequence of humans has been worked out, and there are between 20-30 thousand human protein coding genes. Although this is a big number, most recent estimates have shown that the protein number in human cells actually far exceeds the number of genes. An important question has now become how does the cell bridge the gap in numbers. One important way seems to be to use the same gene to encode more than one protein. The 1993 Nobel Prize was awarded for the important discovery that the genes of organisms like humans are split between bits that encode proteins (called exons) separated by non-coding regions (called introns). DNA is copied into RNA which in turn is used to make protein. After RNA is made, exons are joined together in the cell, by removing introns to give the template which encodes protein. Frequently different exon combinations are included into RNA from the same gene, resulting in variation. For instance, sometimes some of the exons are removed along with the introns. This process (called alternative splicing) is critically important in development, and might even have been an important evolutionary step in allowing the development of multicellularity. Despite this, it has not been studied as much as the controls (transcription) which decide which genes are turned on and off to make the RNA in the first place. Alternative RNA splicing is controlled by proteins which bind to RNA in the nucleus. Some of these proteins are not made in every part of the body but only in particular tissues such as the brain or the testis. Evidence so far suggests that these are likely to have very important roles. One of these RNA binding proteins, called T-STAR, is of particular interest since it may play roles in splicing and connecting signalling pathways with RNA processing and possibly even transcription during development. A good way of investigating the function of a human gene is to look at the equivalent mouse gene, and we propose to test the role of T-STAR in mouse development. Mice, like humans, have a T-STAR gene which is turned on in the adult testis, developing brain and kidney. Although mouse and human T-STAR proteins are virtually identical, they have an important difference in that they are regulated differently. For this reason we predict that T-STAR protein will regulate the same genes differently in humans and mice, and this might help explain some of the reasons mice are different from humans. We will make a conditional version of the T-STAR gene in mice. Next, we can inactivate this conditional T-STAR by cutting an important part of it out of the chromosome when we want to. The way we do this is by mating the mice with special mice which express another protein called a 'recombinase'. We will first remove T-STAR from every cell in the mouse body by turning on the recombinase in every cell. We expect to see defects in the brain, kidney and germ cells,but it could be that these mice will die while they are developing . For this reason, we will also selectively remove T-STAR in the testis (where the sperm are made). This is the main site of T-STAR expression in the adult, and it is an unusual organ because you can see all the major stages of sperm development occurring in the adult and it is non-essential. The mice might be infertile but they will not die. Hence by removing T-STAR in this tissue, even if we get a block in development we will be able to analyse this in the adult mouse and obtain mutant cells. We will analyse gene expression in these mice to see if different genes are expressed, and if transcripts contain different exons from mice which do not contain the deletion. To find out if these mouse genes are regulated differently in humans, we will then compare transcription and splicing patterns of genes affected by T-STAR deletion in the mouse with their human counterparts..