Research
The export of mRNA from the nucleus to the cytoplasm is a critical step along the pathway of gene expression. mRNAs leave the nucleus through nuclear pore complexes (NPCs), and over the past 10 years, studies from this and other labs identified the ~30 proteins which are found in NPCs and many soluble factors that also play roles in mRNA export. Today, the field is focused on understanding how these different proteins work together. Some package the mRNA into RNA/protein complexes; others mediate interactions between these complexes and NPCs. Others participate in a surveillance process to prevent export of any mRNAs that are incompletely or inaccurately processed or packaged. Another major focus within this field is how nuclear pore complexes are assembled to make a structure forty times the size of a ribosome using far fewer components.
- One focus of the lab is Dbp5, a DEAD-box protein. Cells contain more than 30 DEAD-box proteins (DBPs) and one or more participate in each step of RNA metabolism. This enzyme plays a key role during translocation of the mRNA through NPCs and we are using a combination of approaches to define the exact steps that occur during translocation and the roles Dbp5 plays in them. The protein also functions in the nucleus and cytoplasm. We are using a set of biochemical assays and dominant negative mutants to define the Dbp5 cycle and the mechanism by which Dbp5 mediates mRNA export. Although some proteins that interact with Dbp5 have been identified, the analysis is far from complete We are taking a proteomic/mass spectrometry approach to identify additional proteins that interact with Dbp5 and this will be followed up by exploring the functional and genetic connections between these proteins and Dbp5. Recently, we have been using fluorescence recovery after photobleaching (FRAP) to examine the dynamics of Dbp5 at NPCs.
- A second focus is assembly and function of the nuclear pore complex (NPC) and the role of the nuclear envelope in NPC biogenesis. Through a genetic screen, we identified a protein important for making new NPCs. This protein, Apq12p, and another to which it is genetically linked, Brr6p, appear to be needed for cells to maintain proper membrane fluidity and flexibility as they construct the NPC, a protein-lined membrane tunnel through the two membranes of the nuclear envelope. Preliminary evidence suggests that Apq12, Brr6, and another protein may function as part of the sensing mechanism by which cells monitor the biophysical properties of their membranes. While it is known that cells have mechanisms to sense membrane properties and induce changes in membrane composition in order to maintain proper membrane behavior, very little is known about how what the sensing mechanism is and how it functions to trigger membrane modifications.
All of our studies use genetic, cytological, and biochemical approaches. We make extensive use of mutants and genetic screens but are also addressing important questions using purified proteins. We use fluorescence microscopy of living cells to examine the behavior of key nuclear transport proteins tagged with GFP and other fluorescent proteins.