Thursday, May 2, 2024
5/2/2024
We have recently uncovered a previously unknown process that enhances the translation of specific mRNAs through the formation of a biological condensate. Many of the granules identified to date contain a mixture of nucleic acids and proteins. In the cytoplasm, often the condensates are ribonucleoprotein (RNP) granules. Two of the best studied of these, the stress granule (SG) and the Processing body (P-body) sequester RNAs and are associated with the inhibition of protein synthesis. In contrast to these previously identified cytoplasmic granules this new biomolecular condensate (5B granule) is associated with the stimulation of translation of specific mRNAs. Stress inducing agents, such as sodium arsenite treatment or sorbitol, stimulate the formation of 5B granules. Interestingly, these same treatments stimulate SG formation and increase the number of P-bodies, suggesting that under stress conditions the cell contains both repressive and stimulatory granules. Under these conditions, and in contrast to stress granules, the 5B granules stimulate IRES activity and represent a new cytoplasmic condensate. In order to understand and start to explore what these granules are we need to know what components are in the granules and how much these granules influence global translation. To this end we are proposing two specific aims. Aim 1. Identify the components of the 5B granule and determine the relationship to other cytoplasmic granules. We will use an unbiased approach and a candidate approach to identify the protein components of the 5B granule. We will use quantitative mass spectrometry to identify proteins in proximity with EIF5B under conditions that favor 5B granule formation. We will use proximity labeling and precipitation to enrich EIF5B associated proteins. We will also take a candidate approach, using markers of the other granules and cytoplasmic RNA binding proteins to determine the relationship of the stress granule, the P-body and the 5B granule. Aim 2. Determine the global effect of the 5B granule on the translation We will use mRNA-seq, polysome profiling, and ribosome protected fragment (RPF) sequencing to determine the extent and specificity of the role of the 5B granule. We have created gain of function and loss of function models for EIF5B. In addition, we have identified the regions of eIF5B that promote granule formation allowing us to control granule formation and assay global effects on translation.
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