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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Nuclear export and cytoplasmic maturation of the large ribosomal subunit

Lo, Kai-Yin, 1978- 24 March 2011 (has links)
The work in this thesis addresses the general problem of how ribosomal subunits are exported from the nucleus to mature in the cytoplasm. There are three parts in this dissertation. In the first part, I asked questions about the specificity for export receptors in the nuclear export of the large (60S) ribosomal subunit in yeast. In principle, I tethered different export receptors that are known to work in various unrelated export pathways to the ribosome by fusing them to the trans-acting factor Nmd3. Interestingly, all the chimeric receptors were able to support export, although to different degrees. Moreover, 60S export driven by these chimeric receptors was independent of Crm1, an export receptor that is essential for 60S export in wild-type cells. The second question I addressed in this project was whether or not a nuclear export signal could be provided in cis on ribosomal proteins (Rpls) rather than in trans by a transacting factor. The nuclear export signal (NES) of Nmd3 was fused to different ribosomal proteins and tested for support of 60S export. Several Rpl-NES fusion constructs worked to promote 60S export. Rpl3 gave the best efficiency. In conclusion, these results imply unexpected flexibility in the 60S export pathway. This may help explain how different export receptors could have evolved in different eukaryotic lineages. In the second part of my thesis, I identified the assembly pathway for the base of the ribosome stalk. The stalk is an important functional domain of the large ribosomal subunit because of its requirement for interaction with translation factors. Mrt4 is a nuclear paralog of P0, which is an essential part of the stalk. Here, I identified Yvh1 a novel ribosome biogenesis factor that is required for the release of Mrt4. Yvh1 is a conserved dual phosphatase, but the C-terminal zinc-binding domain rather than the phosphatase function was required for its activity to release Mrt4. Mrt4 localizes in the nucleus and nucleolus in the wild-type cells, but was persistent on cytoplasmic 60S subunits in yvh1[Delta] cells. The persistence of Mrt4 on the 60S subunits blocked the loading of P0 and assembly of the stalk. I also found the binding of Yvh1 depended on Rpl12, a protein that binds together with P0 to form the base of the stalk. Deletion of Rpl12 phenocopied yvh1[Delta]. These data identified the function of Yvh1 as a release factor of Mrt4. I also showed that the function of Yvh1 is conserved in human cells. In my final project, I analyzed the interdependence and order of the known cytoplasmic maturation events of the 60S subunit. 60S subunits require several maturation steps in the cytoplasm before they become competent in translation. There are four major steps involving two ATPases, Drg1 and Ssa1, and two GTPases, Efl1 and Lsg1. In my study, I ordered these steps into one serial pathway. Drg1 releases Rlp24 in the earliest step of 60S maturation in the cytoplasm. Truncation of the C-terminus of Rlp24 blocked cytoplasmic maturation of the large subunit by preventing the recruitment of Drg1 and led to a secondary defect in the release of Arx1 because of a failure to recruit Rei1. Deletion of REI1 mislocalized Tif6 from the nucleus and nucleolus to the cytoplasm and deletion of ARX1 suppressed the Tif6 mislocalization, indicating that the release of Arx1 was required for Tif6 release downstream. I found that mutation of efl1 or sdo1, the known release factors for Tif6, also blocked Nmd3 release. Tif6-V192F, which could bypass the growth defects of efl1 or sdo1 mutants, suppressed the defect of Nmd3 recycling. These results showed that the release of Tif6 was a prerequisite for Nmd3 release. Thus, the release of Nmd3 is downstream of the Tif6 release step. In conclusion, I have ordered the events of cytoplasmic maturation with Drg1 as the first step after ribosome export, followed by Rei1/Jji1 and then Sdo1/Efl1. The release of Nmd3 by Lsg1 appears to be the last step of ribosome maturation in the cytoplasm. Thus, the two ATPases Drg1 and Ssa work first and then the two GTPases Efl1 and Lsg1 work in a linear pathway of 60S maturation in the cytoplasm. / text
2

Regulation of nuclear tRNA export in response to nutrient stress is not evolutionarily conserved and requires the TORC1 and PKA signaling pathways in Saccharomyces cerevisiae

Pierce, Jacqueline 18 January 2013 (has links)
Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability.

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