What causes synonymous codon-specific ribosome fall-off?
In many cases, limitation for an amino acid causes ribosome stalling and fall-off that are completely restricted to a subset of the synonymous codons that encode the amino acid. In other words, translation experiences a shortage of only a specific charged isoacceptor tRNA, not simply the limiting amino acid. This creates a powerful mechanism whereby protein sequence-neutral evolution of codon usage can sensitize or desensitize protein production to amino acid levels. We use charged tRNA-seq and other biochemical approaches to study the asymmetrical effects of amino acid starvation on tRNA charging, levels, and modification state to understand synonymous codon-specific ribosome fall-off. Other projects examine disease phenotypes associated with amino acid limitation-sensitive or -insensitive codons.
What is the mechanism of tRNA-starved ribosome fall-off?
The quality control mechanisms that catalyze premature ribosome fall-off have come into beautiful biochemical and structural focus across the last decade in the context of mRNA damage, defects, and treatment with drugs that create irreversible obstacles for the ribosome. Alicia’s postdoc work has revealed that ribosome quality control pathways also act on the ribosome on intact mRNAs during amino acid starvation. Using biochemical and sequencing-based methods to study translation and mass spectrometry to characterize metabolism, the Darnell lab explores the idea that charged tRNA limitation may be an important physiological input for ribosome quality control pathways, and that these pathways may operate differently on “starved” ribosomes than on “stuck” ones.
In collaboration with the Chidley lab at Duke, we also combine genome-wide and focused CRISPR interference and activation screens with our fluorescent reporter for amino acid limitation-induced ribosome fall-off to discover the regulators and machinery involved in this process. These projects use a range of cell biological, genetic, and biochemical tools to decipher how new candidates control ribosome fall-off.
How do ribosome stalling and fall-off impact cell function and disease?
In addition to stress and signaling responses, amino acid limitation at the ribosome has been linked to specific biological phenotypes from biofilm formation to tumor metastasis and innervation. In a collaboration with Joey Davis’ lab at MIT, we are using quantitative proteomics to form and test hypotheses about how ribosome fall-off impacts the proteome in these environments. Other projects examine physiological and disease contexts in which the balance between translational demand, adaptive signaling activity, and metabolite availability is primed for a loss of homeostasis leading to ribosome fall-off in order to understand its consequences and potential as a therapeutic target.
Core techniques
As described above, our lab is question-driven and uses a variety of biochemical, genetic, and genomic tools to study protein synthesis in cancer cells across metabolic environments in culture. Central to our approach is a handy fluorescent reporter library that can detect ribosome stalling and fall off at any sense codon during amino acid limitation. We use liquid and gas chromatography/mass spectrometry to measure intracellular amino acid metabolism, charged tRNA sequencing to assess the tRNA pool, and ribosome and polysome profiling to examine protein synthesis. We are always excited to form new collaborations to study protein synthesis across physiological and disease models, or dive deeper into the mechanisms we study – please reach out!
