The advent of deep sequencing has enabled the discovery of a multitude of novel cellular functions for RNA. Even in the face of all of this new knowledge, long-standing, fundamental questions remain about the classical messenger function of RNA: how it mediates gene expression into protein. My research group will use chemical biology to elucidate spatial and structural regulation of mRNA degradation inside living cells as a mechanism of gene expression regulation, to identify sites and cellular roles of non-canonical translation initiation, and to understand the scope and regulation of small open reading frame (smORF) translation across evolution. These methods will provide new insight into how cells turn off gene expression from both normal and disease-related mutant mRNAs, as well as how unexpected translation start sites and smORFs in cellular mRNAs generate proteins that are essential for cell survival during (patho)physiological processes such as apoptosis, cell division, and mutagenic stress.
1. How is mRNA degradation regulated in human cells?
Cytoplasmic mRNA degradation regulates gene expression by controlling mRNA levels and half-life, and eliminates disease-related mRNAs containing premature stop codons. This process may be spatiotemporally regulated in cells by a newly discovered human microprotein, NoBody, which inhibits mRNA decay and associates with ribonucleoprotein particles called P-bodies. Understanding how a ~7 kDa microprotein controls the activity of a massive macromolecular complex, and how it is, in turn, regulated, may reveal new principles of spatiotemporal regulation of mRNA turnover as well as general insights into the functions of microproteins.
2. Non-canonical translation initiation: identifying protein products and cellular roles
The accepted model for eukaryotic translation initiation is that initiation factors recognize the mRNA 5’ cap, and then assemble small ribosomal subunits in initiation complexes that scan the mRNA until they reach the first ATG codon, where translation begins. In reality, ribosome profiling and proteomic studies have revealed that translation from non-canonical start sites, including internal sites and non-ATG start codons, is widespread. We believe that these non-canonical translation events are actually widespread and essential to cellular survival, rather than being rare and aberrant. We will develop methods to isolate and detect products of non-canonical translation initiation, as well as to explore a fundamental question: why would evolution select for such permissivity in translation initiation?
3. Differential expression of small open reading frame-derived microproteins
We and others have applied new technologies for genome reannotation, including mass spectometry-based proteomics and ribosome footprinting coupled to deep sequencing, to the demonstration that thousands of small open reading frames are translated to produce microproteins in both prokaryotic and eukaryotic genomes. However, it remains unclear what fraction of these newly discovered microproteins are functional. We believe that microproteins that exhibit regulated expression or disease-specific expression are likely to be functional. We recently developed quantative approaches to non-annotated microprotein detection in bacteria that led to the discovery of novel E. coli stress response proteins (heat shock and cold shock). These approaches will be of great utility in further exploration of microbial stress responses as well as human disease.