In eukaryotic cells, genes encoded within DNA are found in the nucleus, a compartment that separates genes from the rest of the cell via the nuclear envelope. Gene expression involves multiple, highly regulated events including messenger RNA (mRNA) transcription, processing, export to the cytoplasm, and translation. During transcription, the mRNA is loaded with mRNA-binding proteins that regulate each of these processes, thus influencing gene expression. All transport events between the nucleus and cytoplasm occur through nuclear pore complexes (NPCs), which are large proteinaceous structures embedded within the nuclear envelope. These NPCs provide both an aqueous channel for molecular movement as well as a docking site for binding of factors that participate in the transport process. The mRNA export receptor heterodimer, Mex67-Mtr2, facilitates movement through the NPC by bridging specific interactions between the mRNA and the NPC.
Figure 1: Messenger RNA Export from the Nucleus. Messenger RNAs are highly dynamic with RNA-binding proteins being added and subsequently removed during the export process. Other factors that influence mRNA export are constitutively nuclear whereas some are associated with the exiting mRNP and are subsequently re-imported into the nucleus in a process called shuttling. DEAD-box proteins such as Sub2 and Dbp5 (not pictured), are proposed to facilitate addition and subsequent removal of Mex67-Mtr2 and other mRNA-associated factors to promote nuclear export of the mRNA.
Although key players in the mRNA export process were identified over 10 years ago, the precise biochemical mechanism of transporting an mRNA:protein complex (mRNP) from the nucleus to the cytoplasm is not fully defined. This is largely due to the compositional complexity of mRNPs and the fact that mRNPs are highly dynamic with RNA-binding proteins being added and removed before, during, and immediately after mRNA export (see Figure 1). These compositional changes, collectively termed mRNP remodeling, provide a quality control mechanism for export of only properly assembled, processed mRNPs to the cytoplasm.
DEAD-box Proteins and mRNA Export
One class of enzymes that function in remodeling RNA: protein complexes are the DEAD-box proteins. DEAD-box proteins are evolutionarily conserved RNA-dependent ATPases, which function in all aspects of RNA metabolism from transcription to mRNA decay. Whereas some DEAD-box enzymes function as ATP-dependent RNA helicases, RNA chaperones or RNP remodeling enzymes, most of the ~25 members in budding yeast S. cerevisiae have not been enzymatically characterized. It is currently unknown how DEAD-box proteins select and subsequently disrupt specific RNP targets in vivo. Therefore, the precise role of DEAD-box proteins in mRNA export is not fully understood.
The goal of our laboratory is to define the precise biochemical mechanism of mRNA export by focusing on the role of DEAD-box proteins in this pathway. We aim to define the physiological targets and mode of action for two DEAD-box proteins, Sub2 and Dbp5, with functions essential to mRNA transport. Our laboratory uses the budding yeast Saccharomyces cerevisiae model system, undertaking a multidisciplinary approach through genetics, microscopy and biochemistry to characterize specific mRNP remodeling events both in vitro and in living cells. Currently, we are developing a live cell method to visualize single mRNA transcripts to allow us to study mRNP remodeling at single RNA: protein complex resolution.
DEAD-box Proteins and Human Disease