Defining the dynamics of eukaryotic translation and mRNA decay

About This Grant

Project Summary / Abstract Protein synthesis, also known a translation, is a highly regulated process with deep significance to public health. Dysregulation of translation is a major driver of diseases such as cancer, and mutations that prematurely halt translation cause 11% of all heritable human diseases. Despite its importance, we do not yet understand the molecular events used to evaluate messenger RNAs (mRNAs) during translation, which serves as a crucial branchpoint in cells. When translation concludes on a normal mRNA, the molecular machinery that drives translation in cells (ribosomes) is typically released from the mRNA to facilitate further rounds of protein synthesis. Yet, for reasons that remain unclear, translation of an aberrant mRNA instead recruits specialized machinery to the ribosome to halt translation and destroy the faulty mRNA. Although we understand that the concluding stages of translation play an outsized role in determining mRNA fate, these branchpoints have proven extremely difficult to study due to the intricate and transient nature of the underlying interactions. As a postdoctoral fellow, I used an in vitro-reconstituted yeast translation system composed solely of purified components to recapitulate translation termination, the process through which newly synthesized proteins are typically released from ribosomes. By attaching fluorescent dyes to the key cofactors that drive translation termination, I was able to watch this process unfold in real time using single-molecule fluorescence spectroscopy to obtain detailed, time-resolved movies of this core cellular process. During the ESI MIRA phase, my research group will apply in vitro systems, traditional biochemical techniques, single-molecule fluorescence spectroscopy, and structural approaches to determine how translation concludes on normal and aberrant mRNAs. In Area 1, we will uncover how translation termination is regulated by local mRNA sequence and proximity of the poly(A) tail to help ribosomes distinguish normal from aberrant mRNAs. We will also determine how recycling, the process through which ribosomes are removed from normal mRNAs after translation, is choreographed. In Area 2, we will characterize the interplay of two interrelated processes (the No-Go Decay and Ribosome Quality Trigger pathways) that are both activated by the collision of ribosomes during translation of aberrant mRNAs. Put together, our investigations will uncover the key molecular interactions that dictate the fate of mRNAs during translation. A detailed understanding of these fundamental gene expression processes could also uncover new ways to manipulate them for therapeutic benefit, paving the way to new treatments for cystic fibrosis, muscular dystrophy, Alzheimer’s, and cancer.