Dissecting the roles of ubiquitin in translation control

研究战略

RESEARCH STRATEGY NIGMS administrative equipment supplement for MIRA GM 1R35GM137954 PI: Gustavo Silva - Dissecting the roles of ubiquitin in translation control I am requesting funds for the acquisition of a multicolor flow cytometer (BD FACSCelesta) capable of quantifying molecular fluorescence at the cellular level. Aligned with the goals of this award, our lab has made great strides in understanding the role of protein ubiquitination in the regulation of gene expression at the translational level. Our recent fortuitous discoveries on sequence-specific translation pausing and mitochondria physiology opened up unexpected opportunities to further advance the goals within the scope of this research program. However, we currently lack instrumentation to perform assays that will elucidate the mechanisms by which ubiquitination of ribosomes controls translation and its arrest under stress. These new capabilities will allow us to advance the goals of this award, providing faster, quantitative, and high throughput data to dissect the roles of ubiquitin in translation control and beyond. Scientific Justification The main goal of our R35 parental proposal is to understand the role of protein ubiquitination in translation control, particular during stress conditions. This award was based on our discovery of a new pathway in which K63 ubiquitination regulates translation in response to oxidative stress. Recently, we have shown that K63-linked polyubiquitin accumulates rapidly in ribosomal proteins after stress induction and controls the elongation stage of translation (Zhou et al., PNAS 2020). We also identified that ribosome ubiquitination is regulated by a triad of enzymes (Ubp2, Rad6, and Bre1), however, the mechanisms by which ribosome ubiquitination impacts translation remains completely unknown. During the development of this R35 award, we have discovered that the ubiquitin conjugating enzyme Rad6 is critical for the regulation of protein synthesis, mitochondria physiology, and cellular resistance to a variety of stresses (manuscript in the final stages of revision in Cell Reports, IF=9.4). Using state-of-the-art ribosome profiling methods, we observed that ribosomes from wild-type cells show a strong pausing (arrest) effect on selective tripeptide motifs under oxidative stress. Impressively, these redox pausing signatures are completely abolished in yeast cells lacking RAD6. Moreover, cells deleted for Rad6 show dysregulation of mitochondria physiology and of the integrated stress response, an important pathway that represses translation globally at the initiation level during environmental stresses and nutrient starvation. These findings are the backbone of a new manuscript currently under preparation exploring the role of Rad6 and ribosomes ubiquitination in translational control. From these discoveries, two critical questions arose in our research program: 1. What is the mechanism by which ubiquitin induces redox pausing signatures under stress? 2. What is the mechanism by which RAD6- deleted cells bypass redox pausing sequences and sustain translation without the activation of the integrated stress response? To address these mechanistic questions, we are developing fluorescent reporter systems (GFP-X-RFP), in which we can test the effect of mRNA sequences in translation elongation. In these reports, “X” can be replaced by unique RNA sequences to determine protein expression, pausing, frameshift, mis- incorporation, codon-specificity, and the mRNA structural context needed for them to occur. Unfortunately, we lack instrumentation capable of quantifying the levels of protein expression to determine the mechanistic rules governing translation under stress. We currently rely on western blots to evaluate protein levels, which is laborious, imprecise (semi-quantitative at best), and only allows a few conditions (strains, conditions, sequences) to be measured simultaneously. The acquisition of a multicolor flow cytometer will allow us to quantify protein synthesis using an array of fluorescent reports in a fast, reliable, and quantitative fashion. Moreover, we do not have access to facilities for radioactivity work with scintillators to measure global protein synthesis using 35S-Met incorporation. Therefore, this flow cytometer will allow us to quantify global protein synthesis through fluorescent Click-it chemistry, in addition to monitor mitochondria abundance and function, quantify reactive oxygen species, and cellular division and viability through the use of several fluorescent dyes. Administrative Justification When we submitted the proposal for the funded parent award, we did not anticipate that we would be using these dual fluorescent reports to determine protein production at the cellular level. Our science is moving in a very fast and in exciting pace, allowing us to discover and explore several new avenues within the scope of our research program. We want to be able to fully solidify our research program, sustain our productivity, and continue to generate high impact contributions to field. This BD FACSCelesta flow cytometer would allow us to keep producing relevant and quantitative data in a high-throughput fashion. We tested and optimized the use of these reporters with flow cytometry but unfortunately there is only one instrument with the laser capability required for our project in the Duke flow cytometry core at the School of Medicine. This is instrument is highly booked because it serves the entire institution, have limited hours of use, it is relatively far from our lab in the main campus, and requires an hourly fee for its use. Another challenging aspect is that the instrument is connected to the only computer that has the proprietary software (FACSDiva) for data analysis. Therefore, we also need to make additional reservations and pay fees for data analysis. We have an increasing demand for this instrument in many fronts of our proposed work and having a freely accessible flow cytometer in Biology will propel our science forward. This is a low maintenance equipment and our operation budget will cover the costs of its upkeeping and usage. Equipment capabilities We propose to use these administrative funds to purchase a BD FACSCelesta benchtop flow cytometer. This is a is a multi-laser, multi-detector instrument that will allow to quantify protein production (GFP, RPF, among others), newly synthesized protein (Alexa-fluor), cell cycle (SYTOX), cell viability (SYTO9), Mitochondria (MitoTracker), ROS (CellRox), among many other cellular features. This high sensitivity, high performance flow cytometer will be equipped with three lasers (blue, 488nm; violet, 405nm; and yellow-green, 561nm), which allow us to measure up to 14 unique parameters in a single sample. Moreover, the BDFACSCelesta is compatible and optimized to work with BD horizon brilliant dyes, a unique set of fluorophores developed for the detection of low abundance species. Finally, the instrument is equipped with BD FACSDiva software that allows a streamlined workflow from acquisition to data analysis, with a range of automated calculations and scaling factors for an improved outcome. Training for use This is a ready-to-use equipment and members of my lab are already knowledgeable in the use of the equipment and its software, trained by members of the Duke Flow Cytometry core. Formal training will be offered by BD during installation of the FACSCelesta, which is already included in the purchase package. Additionally, because of its worldwide usage, there is a large array of resources and tutorials online for sample processing and data analysis. Additional Commitment This is a low-maintenance equipment and funds from our operation budget will be allocated to support the purchase of reagents for regular use, maintenance, cleaning, and sterilization of the instrument. Availability of equipment to NIGMS-funded labs in the Department of Biology. Although several students and trainees in the Silva lab supported by this award will heavily use this equipment, there are additional NIGMS-funded labs in the Department of Biology at Duke that will have access and benefit from this instrument to further their research goals. These groups also rely on the core facility and it will improve their accessibility to this instrumentation. Among the labs that have already expressed interest in having a flow cytometer in the department: Dr. Lucia Strader (R35GM136338) Dr. Philip Benfey (R35GM131725) Dr. Dave Sherwood (R35GM118049)