Date of Award
Mary J. Clancy
One lesser-known but universal post transcriptional modification carried out in yeast and higher eukaryotes is the methylation of mRNA, as mediated by the Ime4 protein and its orthologs. Ime4 protein is essential for sporulation in yeast cells and for viability of higher eukaryotic cells. The precise locations of the Ime4 protein and the functions of the methylated mRNA are still largely unknown. Whereas Ime4 protein is believed to be exclusively nuclear in higher eukaryotes, we have observed the yeast Ime4 protein in the nucleus, in the cytosol and within cytosolic particles. These observations suggest that Ime4 could be a shuttling RNA binding protein, playing roles in the cytosol as well as the nucleus. As a first step to examining this idea, we tested the hypothesis that the punctuate cytosolic particles formed by Ime4 are P bodies. P bodies are transient aggregates of proteins and RNAs that form as a result of stresses such as glucose deprivation. This experiment was carried out using fluorescence microscopy using Ime4 tagged with GFP (green fluorescent protein) and the known P -body proteins Edc3, tagged with mCherry. We expected that if the proteins thus produced localized in the same place in the yeast cell, we could then deduce that Ime4 is present in P-bodies. We observed that Ime4 and Edc3 did not colocalize in the majority of cells, and thus concluded that the Ime4 granules are not P-bodies. However, our experiments showed instances of Ime4 signals near or around the P-bodies in some cells. Hence, the Ime4-containing aggregates are not likely to be P-bodies but could rather represent a different type of granule.
Ghimire, Jenisha, "Localization of Ime4 in Saccharomyces cerevisiae" (2012). Senior Honors Theses. 12.