LOS1P INDEPENDENT NUCLEAR TRNA EXPORT IN SACCHAROMYCES CEREVISIAE A ROLE FOR NUCLEAR TRNA AMINOACYLATION AND EXPORT

Open Access
- Author:
- Eisaman, Duane
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 02, 2004
- Committee Members:
- Anitia Hopper, Committee Chair/Co-Chair
Hui Ling Chiang, Committee Member
Kristin Ann Eckert, Committee Member
Thomas Wright Gardner, Committee Member
Ralph Lauren Keil, Committee Member - Keywords:
- tRNA
yeast
RNAi
Export
cerevisiae
Sol1 - Abstract:
- Proper movement of macromolecules between the nucleus and the cytoplasm is essential for life. The transport of molecules is an energy-dependent and receptor-mediated process. All transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). The export of tRNA from the nucleus, where it is produced, to the cytosol, where it functions in translation, is also an essential process. The only Ran GTP binding receptors that has been shown to bind tRNA in yeast is Los1p. The LOS1 gene is unessential in yeast. Therefore, there must be alternative pathways that allow the tRNA to move out of the nucleus into the cytosol. There is strong evidence that aminoacylation of tRNA may be involved in such pathways. Previous research has shown that aminoacylation or charging of tRNA is important for efficient nuclear export of tRNA. Temperature sensitive mutations of the aminoacyl-tRNA synthetases resulted in nuclear accumulation of tRNA in yeast. In addition, if the nuclear pool of tyrosyl-tRNA synthetase was reduced in the cell, the cell accumulated tyrosyl-tRNA in the nucleus. Overexpression of methionyl-tRNA synthetase in los1ƒ´ strain corrected the nuclear accumulation of methionyl-tRNA. Mutation of CCA1, an enzyme that adds the acceptor arm to the tRNA, a necessary first step in the charging of tRNAs, resulted in nuclear accumulation of tRNA. In addition, over expression of CCA1 in los1ƒ´ strains resulted in correction of the nuclear accumulation of a majority of tRNAs. Overall, these results strongly indicated that aminoacylation was critical for tRNA transport. A biochemical screen of proteins implicated in tRNA nuclear export was initiated to elucidate the mechanism of aminoacylation in tRNA nuclear egress. By examining the protein-protein and tRNA-protein interactions in nuclear tRNA transport, a better understanding of nuclear tRNA export is possible. Gst-tagged versions of the two aminoacyl-tRNA synthetases, TYS1 and ILS1, were found to complement their respective temperature sensitive mutants. Each protein was purified and was able to aminoacylate tRNA in vitro. Both proteins co-purified with their cognate tRNAs. The tRNAs were released in high salt. Interestingly, Gst-Tys1p also co-purified with Gat4p, a possible nitrogen-regulated transcription factor and endogenous Tys1p. Gst-Ils1p did not co-purify with any other proteins. Exhaustive attempts were made to identify components of the tRNA transport/maturation pathway by searching for proteins that co-purified with Tys1p and Ils1p, but no known components of this pathway were found. An alternative approach with aminoacyl-tRNA synthetases, MES1 and mes1-1 was taken to reach the same goal. The Mes1-1p has a single amino acid substitution and previous work demonstrated temperature sensitive growth as well as tRNA nuclear accumulation at the nonpermissive temperature. The temperature sensitive growth phenotype was restored by the addition of methionine to the media and published data showed that the mutant protein has 100-fold less binding affinity for methionine as compared to Mes1p, suggesting Mes1-1p is compromised in aminoacylation activity. If Mes1-1p is only defective in aminoacylation and has normal tRNA binding, the tRNA nuclear accumulation phenotype could be due to a defect in aminoacylation rather than tRNA binding. To test this hypothesis, I attempted to compare the tRNA binding affinity between Gst-Mes1p and Gst-Mes1-1p. It was determined that Gst-Mes1-1p is likely a less stable protein than Gst-Mes1p and therefore a clear conclusion on the role of aminoacylation and tRNA binding could not be made. SOL1 was found to be a multicopy suppressor of los1ƒ´, which suggests that SOL1 may function in a Los1p-independent tRNA export pathway. A functional, tagged version of the gene was constructed and the encoded protein was purified. Gst-Sol1p co-purified with Tef1/2p, the elongation factor 1 alpha. Tef1/2p has been shown to bind aminoacylated tRNA and to facilitate the movement of tRNA into the A site of the ribosome. Even though Tef1/2p was known to bind tRNA, tRNA did not co-purify with Sol1p and Tef1/2p, which suggested this interaction is not tRNA dependent. This newly discovered Sol1p/Tef1p complex might form the basis for an aminoacylation-dependent tRNA export pathway in which Sol1p binds Tef1p importing Tef1p into the nucleus. Tef1p could then bind aminoacylated tRNA, releasing Sol1p with Tef1p moving out of the nucleus carrying tRNA, thereby functioned as an aminoacylation-dependent tRNA nuclear export pathway. RNAs in the eukaryotic cells were originally thought to function as structural components of the protein translation. Recently, RNAs are found to edit RNA sequences and to act in the catalytic roles. Now it appears that RNAs may regulate the expression of the genome and may be integral to the proper development of many multicellular organisms. This process is called double stranded RNA interference (RNAi). RNAi has been found in many different organisms across many eukaryotic families. RNAi is the silencing of mRNA with high sequence homology to a dsRNA initiator molecule. The mechanism and regulation of RNAi is still poorly understood. A strong, easily manipulated genetic model for RNAi would be invaluable to study RNAi. Therefore, we attempted to use Saccharomyces cerevisiae to develop such as model. A plasmid that was expected to express a double stranded RNA was transformed into yeast. A GFP gene under a GAL1 promoter was transformed into the cells. The dsRNA was targeted against the negative regulator of the GAL regulon, GAL80. The dsRNA construct activated the expression of GFP. RNAi was expected, based on all previous published research, to reduce the levels of GAL80 mRNA and produce 21-23 bp RNAs (siRNA) against the GAL80 mRNA. The dsRNA induced expression of GFP in yeast failed to show a reduction of GAL80 mRNAs or proteins. No siRNAs were detected. These data suggested that the dsRNA activation of GFP was not due to classical RNAi, but due to a nonspecific RNAi reaction or another unknown dsRNA activation of the GAL regulon.