Browsing by Subject "Yeast"

Post-transcriptional modifications (PTMs) in RNA are present in all known RNA species and conserved in all kingdoms of life. Transfer RNA (tRNA) has been shown to have numerous conserved modifications, which exemplifies the importance of modifications having impact on the structure of the tRNA and its function as carrier of the amino acids. Ribosomal RNAs (rRNA) are universally modified as well, and modifications are situated at functionally important spots of the ribosome. Given the fact that types and sites of modifications are conserved, it is likely that these modifications have been selected for and that they optimize the ribosomal structure and functions. Stress, such as temperature or infection by a pathogen, is known to change the presence or abundance of modifications in RNA molecules and thereby affect translation efficacy. In line with that, this master’s thesis project sought to gain insight into the dynamics of PTMs in tRNA and rRNA upon oxidative stress, with the goal of utilizing recently optimized UPLC/MS method for identifying modified ribonucleosides. As the specific aim of the thesis was to estimate the change in PTMs in tRNA and rRNA in response to oxidative stress with 0.5 mM and 2 mM hydrogen peroxide H2O2, 3 immediate goals were: (i) to isolate total tRNA from yeast grown in stress conditions, (ii) to isolate rRNA from yeast 80S ribosomes, and (iii) to identify present modifications using mass spectrometry. Yeast was cultured in presence of H2O2 as a stressor in mentioned concentrations, and both treatments considered showed a difference in survival when compared to the control. Rough cell concentration estimates (OD600) did not show the effect of the stressor on cell survival clearly, but when number of viable cells per mL was estimated, it was clear that growth of the stressed yeast cultures was hindered 2 hours after exposure to H2O2 but recovered during the 24 hours. Firstly, using UPLC/MS analysis, 29 modifications were identified in tRNA from control and H2O2 treated yeast. Most identified modifications showed no change in abundance in treatments, which is to be verified with additional replicates. However, distinct dynamics of stress-related change was found for several modifications, revealing additional modifications that may play a role in stress related modificome reprogramming to the previously known signature modifications of oxidative stress. It was expected that recovery of culture growth after 24 hours may be accompanied with modification level recovery. However, that was not demonstrated here as downregulation at 2 hours followed by upregulation at 24 hours was seen for 2-methylthio-N6-methyladenosine, N4-acetylcytidine and 5-methoxycarbonylmethyl-2-thiouridine, and the reverse was shown for N4-methylcytidine. Upregulation in both time points was also shown here for some modifications. Taken together, these results confirm a complex and dynamic control of tRNA modifications in cellular survival responses. Modifications found to be affected by oxidative stress are most frequently located on the wobble position 34 and anticodon loop position 37, so it is expected that changes in their modification levels could directly affect the tRNA function in translation, making them a specific target for future research. Secondly, modifications in rRNA from control yeast cultures were identified, such as expected methylations of all 4 canonical nucleosides. However, further analysis will be needed to confirm the other identified modifications, due to the potential mRNA and tRNA contamination. Optimizing the method for rRNA modifications identifications by acquiring more modified nucleosides specific for the rRNA to use as standards in the analysis, analyzing rRNA types separately and using tandem mass spectrometry would enable getting a deeper understanding of which modifications are present and where they are positioned. Finally, it would enable reliable identification of the signals of novel modifications present in rRNA, such as the tRNA modification 5-carbamoylmethyluridine signal found here. In conclusion, this thesis work lays the foundation to study the evolutionary conserved function of PTM changes during stress as modulators of translation, using the methodological approaches discussed in-depth within the thesis, primarily to confirm the intriguing results found here.

Tiivistelmä – Referat – Abstract The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase is a master regulator of cell growth. In the presence of environmental cues, such as nutrients and growth factor, mTORC1 is transported to the lysosome where it is activated by a small GTPase Rheb. Dysregulation of mTORC1 has been linked to several diseases such as cancer and neurodegeneration. Despite our growing understanding of the nutrient-driven activation mechanism of mTORC1, we still do not fully understand how nutrients are transported out of the lysosome or how nutrient sensing is connected to nutrient transport. Recently, SLC38A9, a small lysosomal transmembrane protein, was identified as a mediator of the efflux of essential amino acids from the lysosome to the cytosol. It also acts as an amino acid sensor for mTORC1, playing a role in its activation. Due to poorly vascularized tumor cores, cancers such as pancreatic ductal adenocarcinoma, have access to very scarce amounts of free nutrients. Consequently, they rely on scavenging of protein macromolecules from the extracellular environment, followed by digestion inside lysosomes. The digested nutrients are released to the cytosol via transporters such as SLC38A9 and activate the mTORC1 pathway which carries out the growth processes. In fact, recent studies in mouse xenograft models have shown a severely slowed down growth of PDAC tumors with SLC38A9 knocked out. Blocking of SLC38A9 activity with pharmacologics or biologics would prevent the release of digested amino acids from the lysosomes, starving cancer cells of nutrients, while sparing normal cells that do not feed on extracellular proteins. However, SLC38A9 is still poorly understood, and development of selective inhibitors first requires mechanistic understanding of the protein and knowing what its binding pockets look like. In order to obtain this information, we aimed to determine the three-dimensional structure of SLC38A9 through cryogenic electron microscopy (cryo-EM). However, two significant challenges hindered our ability to obtain high-resolution images of this membrane protein: (i) its small size, and (ii) its constant conformational changes. To address this, I proceeded to develop a set of nanobodies that would bind SLC38A9 with high affinity and specificity. Nanobodies allow for locking of target proteins in specific conformational states, and they can also serve as chaperones for visualizing proteins in cryo-EM. To obtain these nanobodies, I used a library of 100 million unique nanobodies, displayed on the surface of yeast cells. Specific SLC38A9 binder nanobodies were obtained through multiple rounds of selection and sorting, using decreasing concentrations of fluorescently- labeled SLC38A9. After the final selection round, single colonies were picked and the strength of binding to SLC38A9 was evaluated. High-throughput screening results showed that we were able to obtain specific SLC38A9 binders and that there was variation in binding strength among the selected nanobodies. These nanobodies will enable the determination of the cryo-EM structure of SLC38A9 and also serve as tools to further dissect the function and mechanisms of SLC38A9 in amino-acid efflux from lysosomes to cytosol, providing further insights for the development of novel cancer therapeutics.