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Evolution of structure and function of Kink-turn containing RNAs in the Domain Archaea

Creative Commons 'BY' version 4.0 license

The Domain Archaea is comprised of prokaryotic organisms known for often living in extreme environments. Despite potential biotechnological uses and their importance to the carbon and nitrogen cycles, archaea are understudied compared to eukaryotes and bacteria. Considering that archaea share features of bacterial RNA regulation (e.g., CRISPRs) and have transcription and translation machinery similar to eukaryotes, elucidating mechanisms of archaeal RNA biology may help us understand the evolution of RNA in the tree of life. For my dissertation I studied archaeal RNAs that contain the kink-turn, an RNA structural motif, with an emphasis on C/D box sRNAs and H/ACA box sRNAs.

I used high-throughput RNA sequencing data and RNA bioinformatics to (1) improve detection and explore biological roles of archaeal C/D box sRNAs, (2) improve detection and analysis of H/ACA box sRNAs across the archaea, and (3) survey kink-turn-containing RNAs in archaea. C/D box and H/ACA box sRNAs guide the 2'-O-methylation and pseudouridylation, respectively, of transfer and ribosomal RNAs in archaea and are likely under-annotated. To improve computational detection of C/D box and H/ACA box sRNAs, I incorporated the RNA kink-turn motif into covariance models. My results are supported by comparative genomics, promoter predictions, and small RNA sequencing from 20 diverse archaeal species. I increased the annotations across the archaeal domain and, in particular, increased annotations by 20-30% in the hyperthermophilic species. I also demonstrated that non-canonical forms of H/ACA box sRNAs are not unique to the Pyrobaculum genus, but are widespread across the archaeal domain and compose approximately half of all H/ACA box sRNAs.

During my work with C/D box sRNAs, I found that the sequences comprising archaeal kink-turns are likely more diverse than previously thought. The kink-turn motif is a three-dimensional RNA motif ubiquitous across the three domains of life and is a key component of important ribonucleoprotein (RNP) complexes, such as the ribosome, riboswitches, and small nucleolar RNPs. Recent discoveries of kink-turns in archaeal RNase P and the unusual form of Pyrobaculum H/ACA box sRNAs indicate that there may be kink-turns in RNAs that we have not yet discovered. To improve understanding of this motif, provide data for computational models, and find novel kink-turn containing RNAs, I sequenced RNAs co-immunoprecipitated with the archaeal kink-turn binding protein L7Ae, which has a broad-binding specificity. I used a strain of Thermococcus kodakarensis with a HA-tagged L7Ae for these experiments. I found evidence that kink-turns occur in mRNAs and some annotated non-coding RNAs.

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