'How is PtdIns(4,5)P2, a membrane lipid messenger, localised and regulated in splicing speckles, a membrane less compartment within the nucleus?

DNA is the code for human life that makes up genes that are used to produce proteins. Proteins are the molecular workhorses that are used by cells to carry out specific functions. Cells in our bodies are constantly under attack from stressors which lead to their damage and demise. For example, many different environmental exposures lead to damage of our DNA. This can include sunlight or harmful chemicals in the air. DNA is the building blocks of our cells, and if it becomes damaged the cell cannot function properly and this can either lead to the cell dying, or often can lead to the development of diseases such as cancer. DNA is contained in a specialised compartment of the cells called the nucleus. To respond to these stressors that damage our DNA, cells have to be made aware of, or sense, the damage and then have to initiate an appropriate response. There is a highly sophisticated machinery in cells that can sense damaged DNA, and when it does so sends an appropriate signal to inform the cell to change its behaviour and respond to it. One of these signals is made up of a family of lipids or fat molecules collectively called phosphoinositides or PPIns for short. Because of their chemical composition these fat molecules like to stay together with other fat molecules which normally forms a membrane, rather like a soap bubble. In fact, the membrane is what forms the outside of a cell. However, in the nucleus surprisingly, these PPIns molecules sit in a specialised place called a splicing speckle which are known not to have any membranes. How PPIns arrive at the speckles and how they are kept there is a mystery which we are now beginning to understand. In the nucleus these speckles are involved in a rather special function, called splicing, that helps the cell to use the DNA to produce proteins. The DNA instruction manual has a peculiar structure. It contains regions called genes which code for proteins. These genes are made up of smaller blocks of DNA; some of which code for part of the protein and are called exons, and other parts between the exons that contain nonsense code called introns. The instructions to make a protein rely on piecing together the coding exons of the DNA while removing the introns. To do this without losing the cells copy of DNA, it is first copied into a similar molecule called RNA, which contains the exons and introns. The nonsense introns are then removed and this process is called splicing. Once the introns have been removed, the RNA can be used to make proteins to help the cell to respond to the stressors. We think that these PPIns are a key part of the whole process. In response to stressors it seems that the amount of these PPIns at the speckle goes up. Remarkably these PPIns have an ability to be able to attract and talk to special proteins and change where they are in the cell and how well they carry out their functions. It turns out that in fact, they bind and talk to many of the proteins that are involved in splicing. Part of this study will work out exactly how DNA damage changes the amount of PPIns at the speckle, and which splicing proteins respond to the increase in PPIns. What this leaves out is the mystery about how the PPIns arrive and stay in the speckle. In a beautifully coordinated manner, we have found that one of the proteins that is involved in splicing, called SRSF2, is able to bind to PPIns and is critical for bringing the PPIns to the speckle and holding them there. How SRSF2 does this will form a major part of this study.
How well splicing works is fundamental to life itself and during human life splicing ability changes, Moreover, SRSF2 is often mutated in blood cancers. PPIns are made and removed by a family of proteins called enzymes and we hope to make drug like molecules that inhibit them. These could be used to specifically control the levels of PPIns in the nucleus; which could then be used to treat several diseases such as cancer and perhaps help during ageing.

Polyphosphoinositides (PPIns) are a family of membrane loving phospholipids that are context dependent messenger molecules. They are present in the nucleus and regulate nuclear processes by interacting and recruiting receptor proteins including epigenetic signalling regulators, splicing and RNA binding factors, transcription factors, and histones. Surprisingly, a nuclear PPIns, PtdIns(4,5)P2, is localised in subnuclear membrane-less splicing speckles. How these membrane loving lipids are brought and maintained at these speckles is unknown and is a burning question in the field. Using affinity chromatography we have found that the RNA recognition motif (RRM) is a new PPIns interacting domain. RRM domains are present in over 500 proteins and are abundant in the SR family of proteins that are intimately involved in splicing and localise to the splicing speckle. One of these, SRSF2, interacts with nuclear PtdIns(4,5)P2 and controls PtdIns(4,5)P2 localisation in the splicing speckle. Moreover, SRSF2 depletion not only reduces speckle-associated PtdIns(4,5)P2, but causes a dramatic accumulation of PtdIns(4,5)P2 in the cytoplasm. These data identify SRSF2 as a critical regulator of PtdIns(4,5)P2 localisation to a membrane-less organelle, and goes against the adopted paradigm that Phosphatidylinositol is bought to the nucleus to initiate nuclear PPIns signalling. DNA damage signalling is known to impact on splicing through regulating SR proteins like SRSF2. In concert, DNA damage signalling also increases the levels of nuclear PtdIns(4,5)P2 associated with speckles. We have discovered another RRM domain containing protein whose localisation at speckles is dependent on the presence of PtdIns(4,5)P2 at the speckle. In this study we will investigate how PtdIns(4,5)P2 is regulated at the speckle and how PtdIns(4,5)P2, in response to stress, such as DNA damage, crosstalks back to RRM-domain containing proteins, in speckles, to control splicing and transcriptional output.