UBC Theses and Dissertations
Non-protein coding RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus Auxilio, Maria
The archaeal L7Ae protein is an integral component of three functionally distinct macromolecular ribonucleoprotein complexes: the 50S large ribosomal subunit, the C/D box modification particles and the H/ACA box particles To better understand the function of the L7Ae protein and to investigate the diversity of RNAs specifically associated with this protein, immuno-affinity chromatography was used to isolate the sRNAs associated with L7Ae and RT-PCR was employed to construct a cDNA library. The isolated sRNAs were divided in different groups based on the presence of common known sequence and structural motifs and/or genomic location. Group one contained six RNAs that exhibited the, features characteristic of the canonical C/D box archaeal sRNAs and one RNA representative of the archaeal H/ACA sRNA family. Group two contained fourteen sequences that were encoded either within, or overlapping the 5' end or 3' end of ORFs mostly coding for transposases. Interestingly, one of the clones in this group corresponded to the 5'-untranslated region (UTR) of L7Ae mRNA, indicating that L7Ae protein is able to interact with its own mRNA. The relevance of this interaction for the expression of L7Ae protein was further analyzed using a S. solfataricus in vitro translation system. Group three contained three sequences form intergenic regions. Group four contained five antisense sequences complementary to the 5' end, 3' end or internal regions within annotated open-reading frames (ORFs) and two sequences antisense to bona-fide C/D box sRNAs. Group five contained two sequences corresponding to internal regions of 7S RNA of the signal recognition particle (SRP). Additionally, in the aim to better understand the versatility of L7Ae in the interaction with various sRNA substrates, we introduced mutations in a model C/D box sRNA and monitored the impact of mutation on the protein binding ability and on the methylation function of the sRNA. My data suggest that L7Ae protein might have an important regulatory role in archaeal cells, serving as a primary RNA-binding factor in various complexes with distinct function. In addition, the results obtained in this study set the stage to further characterize all the sequences identified in this screening and to elucidate their function.
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