Nuclear contributions to ASH1 mRNA localization

Abstract

Spatially restricted translation is a means to generate protein asymmetry. One of the best examples of this is illustrated by the budding yeast Saccharomyces cerevisiae, which expresses ASH1 mRNA in late anaphase and localizes it to the bud tip. Ash1p is a transcriptional repressor of the HO endonuclease, important for mating type switching. This localization serves to restrict the production of the ASH1 protein product (Ash1p) to the daughter nucleus. ASH1, as well as at least 23 other bud-localized mRNAs in S. cerevisiae are localized by the cooperative action of cis-acting sequences within the mRNA and trans-acting protein factors. Most of the trans-acting factors assemble on the ASH1 mRNA and remain associated during localization. This localizing mRNP complex is termed the "locasome". Locasome formation is a multi-step process that begins in the nucleus and ends at the bud tip with translation. Though many trans-acting factors have been identified, the process is not understood. For factors that are known, it is still unclear exactly where and when they associate with ASH1. Additionally, new proteins that affect ASH1 localization are still being discovered. Using a method for fluorescently labeling transcripts in living cells, we have imaged a localizing reporter transcript in strains mutant for various trans-acting factors. From this, we determine the contribution of the factors to both locasome formation and localization. One of the mutants, she3 showed a unique phenotype: locasome formation was enhanced, yet localization was still abrogated. In she3 cells, localization-competent transcripts aggregate on the cytoplasmic side of the nuclear envelope. This indicates that post-export, localizing transcripts are retained in proximity to the nuclear periphery until She3p association, which facilitates their detachment from the periphery and directs their localization to the bud tip. This suggests that localization competence is determined prior to export from the nucleus since the assembled locasome is not translated when entering the cytoplasm, but must he inhibited until it reaches the bud tip. We hypothesized that the locasome assembly may be affected by nuclear pore components, since they are composed of proteins associated specifically with the periphery. Selective screening of nucleoporins and other proteins associated with the nuclear periphery reveal that the nuclear basket protein, Nup60p had an effect on the localization of ASH1. Further analysis revealed that in a nup60 strain, not only ASH1 but other localized transcripts were partially retained in the nucleus. Only localized transcripts showed increased nuclear retention in nup60. This indicates that a nucleoporin can regulate a subset of transcripts. Unlike most previously identified trans-acting factors, Nup60p does not remain associated with ASH1 post-export. However, despite not being a component of the locasome, Nup60p is still able to facilitate the localization of ASH1. This reveals the critical nature of nuclear interactions in the localization pathway and identifies a new function for a nuclear pore.