Over one hundred types of chemical modifications have been characterized in cellular RNAs. Pseudouridines (Ψ) and 2’-O-methylation of ribose sugars are the two most widespread modifications present in rRNAs, tRNAs and snRNAs. These modifications can be either guide-RNA mediated or RNA-independent (enzyme only). The RNAs that guide pseudouridylations are called box H/ACA RNAs and the ones that carry out 2’-O-methylations modifications are called box C/D RNAs. Previously, we identified that sR-h45 is the box H/ACA guide RNA responsible for Ψ1940, 1942 and 2605 formation in 23S rRNA of Haloferax volcanii. This RNA has two stem loops – SL1 and SL2. SL1 acts as the guide for Ψ2605 formation and SL2 is responsible for guiding Ψ1940 and Ψ1942. We found that SL2 sequentially guides Ψ1940 and Ψ1942 formation in the unpaired "UNUN" target. Ψ1942 is produced after and only if Ψ1940 is produced. The requirement for conserved ACA box was determined by using variants of these two stem loops. We found that the ACA motif is not required either in vivo or in vitro for the activity of the typical variants of both SL1 and SL2 but required for the activity of the atypical variants of these guides. Cbf5 is the pseudouridine synthase involved in this box H/ACA RNA guided process. Mutants of Methanocaldococcus jannaschii Cbf5 were used with both typical and atypical guide variants in vitro and certain residues were found to be important only for the atypical reactions.We have also studied sR-h41, which is a unique single guide box C/D guide responsible for methylation of G1934 position of 23S rRNA of Haloferax volcanii. We have done in vitro assembly reactions using mutants of sR-h41 assembled with its cognate proteins from Methanocaldococcus jannaschii to study the structural determinants needed to convert it to a dual guide RNA. The assembly pattern of the core proteins on the conserved box C/D and box C’/D motifs steer the dual guide nature of these archaeal box C/D guide RNAs.Another aim of this study was to determine the role of pseudouridine synthases (Pus enzymes) for Ψ55 formation in mammalian tRNAs. We find that three Pus enzymes – TruB1 (in the nucleus), TruB2 (in the mitochondria) and Pus10 (in the cytoplasm) are responsible for this modification depending on the specific sub-cellular location in the cell. These enzymes exhibit different structural requirement for Ψ55 formation that are located on the TΨC loop of tRNAs. A subset of tRNAs like tRNAs for Trp and Gln are protected from the action of TRUB1 in the nucleus by binding to the nuclear version of Pus10 that lacks Ψ55 activity. Ψ55 in this subset of tRNAs is produced by the cytoplasmic version of Pus10.While studying pseudouridylation functions of Pus10, we also found that Pus10 regulates G1/S cell cycle progression in PC3 cells. It does so by directly repressing another protein c-Rel, that is a positive regulator of Cyclin D1 protein. Cyclin D1 is known to play a central role in transition of cell from G1 to S phase during cell cycle progression. c-Rel also regulates the levels of PUS10 by an unknown mechanism.
| Identifer | oai:union.ndltd.org:siu.edu/oai:opensiuc.lib.siu.edu:dissertations-2789 |
| Date | 01 May 2020 |
| Creators | mukhopadhyay, shaoni |
| Publisher | OpenSIUC |
| Source Sets | Southern Illinois University Carbondale |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations |
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