The control of biological processes, such as cellular growth and differentiation, is dependent on how the genetic material within a cell is expressed. The cellular physiology of mRNA—including mRNA processing, transport, localization, and turnover—is central to the process of gene expression. In my laboratory, we are focusing on understanding how cells regulate the translation, localization, and degradation of mRNAs. In eukaryotic cells the decay rates and the translation rates of individual mRNAs can be quite different, and these processes can be regulated in response to a variety of signals, including specific hormones and viral infection, or as a consequence of differentiation. Our goal is to first understand the molecular mechanisms that control mRNA stability and translation rate in eukaryotic cells, using yeast as a model system, and to then extend that basic understanding to other areas of biology.
Our initial work in yeast led to our description of two major pathways of mRNA decay conserved in eukaryotic cells. Both pathways begin with shortening of the 3' polyadenylate tail found on eukaryotic mRNAs (referred to as deadenylation), which primarily triggers decapping, leading to 5' to 3' exonucleolysis. Alternatively, removal of the 3' polyadenylate tail can expose the mRNA to 3' to 5' degradation. We have identified all the critical enzymes involved in these pathways, and we have worked with Haiwei Song (Institute of Molecular and Cell Biology, Singapore) to solve the high-resolution structure of the decapping enzyme and its regulators.
As we continue to study the process and control of mRNA decapping, we are focused on two key issues. First, to understand this process at high resolution, we aim to determine the structures of higher-order mRNP complexes containing the decapping enzyme, its regulators, and the mRNA substrate. Second, to understand the control of translation and degradation, we are addressing how translation factors and the decapping enzyme compete for the 5' cap structure and how cells regulate this competition to control mRNA translation and degradation.
An additional aspect of the control of cytoplasmic mRNAs is that nontranslating mRNPs in eukaryotic cells assemble into conserved and highly dynamic cytoplasmic mRNP granules known as P-bodies (processing bodies) and stress granules. Stress granules, which are observed when translation initiation is limiting (e.g., during stress responses), consist of mRNAs associated with some translation initiation factors and RNA-binding proteins, and thus are thought to represent a pool of mRNPs stalled in the process of translation initiation. P-bodies, which consist of mRNAs associated with translation repressors and the mRNA decay machinery, can be stored mRNAs or mRNA targeted for degradation.
P-bodies and stress granules are important because they are involved in mRNA degradation, cell signaling, stress responses, translational control, and viral life cycles and can also be a source of mRNAs to return to translation. Moreover, stress granules, and to lesser extent P-bodies, are closely related to mRNP granules in neurons and early embryos that play important roles in the regulation of localized translation. We have shown, in collaboration with Mani Ramaswami (Trinity College Dublin), that components of stress granules and P-bodies are required for neuronal mRNP granule formation and at least some forms of synaptic plasticity in Drosophila. This is consistent with neuronal granules being functionally and biochemically related to stress granules and P-bodies. A key question under investigation in our lab is the composition of mRNP granules during their formation and resolution and how the dynamics of mRNP granules affect the function of specific mRNAs.
We have identified several mechanisms regulating the formation and clearance of mRNP granules. Individual mRNPs assemble into P-bodies or stress granules by dimerization and/or oligomerization domains on RNA-binding proteins, including prion-like domains (amino acid domains that are predicted to have a high probability of being able to form amyloid structures). RNA-binding proteins are enriched in such prion-like domains from yeast to mammals, arguing that the use of these domains to form higher-order assemblies is conserved. We have also shown that stress granules can be both disassembled (presumably by chaperonins) and targeted for autophagy, which provides an additional clearance mechanism. An important unresolved issue is whether there is specificity in which stress granule proteins or mRNAs are disassembled and reutilized and those components targeted for autophagy.
Stress granules are of significant interest because recent observations argue that increased stress granule assembly or persistence can be causative in some degenerative diseases. For example, conditions such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and spinocerebellar ataxia type 2 can result from mutations in known stress granule assembly domains, which often increase their tendency to aggregate.
Strikingly, the role of RNA-binding proteins with prion-like domains in degenerative pathologies may be common, as mutations in 10 of the top 20 human RNA-binding proteins predicted to have prion-like domains (e.g., hnRNPA1, TDP-43, FUS, AXTN1, TAF15, hnRNPA2B1, and TIA1) have been linked to some form of degenerative disease. In collaboration with Paul Taylor's lab (St. Jude Children's Research Hospital), we have shown that pathogenic mutations in the AAA-ATPase valosin-containing protein (VCP) that predispose individuals to inclusion body myopathy inhibit clearance of stress granules. Since enhanced stress granule formation (due to hyperassembly mutations in RNA-binding proteins) and a failure to clear stress granules (due to VCP mutations) both lead to stress granule persistence and inclusion body myopathy, an emerging hypothesis is that increased stress granule persistence or formation contributes to the etiology of these diseases. Consistent with that view, a hallmark of ALS, FTLD, and some other neurodegenerative diseases is the accumulation of cytoplasmic aggregates that contain several stress granule factors (e.g., TDP-43) and RNA. An important unresolved question, which we are addressing in our lab, is how these RNP aggregates seen in pathological conditions are related to normal stress granules and how the RNP aggregates affect the biogenesis and function of cellular mRNAs.
A grant from the National Institutes of Health provided additional support for some of this work.
As of October 1, 2013