Ditte Fogh Heidemann:
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RNAs have a wide range of functions as messengers, amino acid adaptors, ribozymes, and regulators, and play fundamental roles in all organisms. This PhD thesis focuses on the transfer RNAs (tRNAs) and small regulatory RNAs (sRNAs) of Escherichia coli (E. coli).
We investigated the regulatory patterns of tRNAs and sRNAs in the context of amino acid starvation. While tRNAs exhibited remarkable stability even under amino acid-deprived conditions, sRNAs displayed a wide range of regulated behaviors.
While it is generally presumed that all tRNA species in E. coli have been identified, the presence of many undefined sRNA genes in the same organism is widely acknowledged. To contribute to closing this knowledge gap within the sRNA field, we developed two methods for the purpose and validated the expression of a novel putative sRNA identified by both methods.
RNA modifications have classically been reserved for the ribosomal RNA (rRNA) and tRNA classes in prokaryotes. However, emerging evidence points to 5' RNA capping as a prevalent modification found on prokaryotic messenger RNAs (mRNAs) and sRNAs. We employed the CapZyme-Seq method to identify capped RNAs in E. coli. Intriguingly, we identified capping of several sRNAs and mRNAs that has not previously been reported. Among these, we validated high capping levels of the sRNA CyaR.
Additionally, we explored potential biases while working with RNA in the laboratory. We identified alarming biases in commonly used RNA extraction methods when using them for extracting RNA from stressed E. coli cultures.
Conclusively, this thesis advances our knowledge within the field of untranslated RNAs of E. coli by developing methodologies for identification of putative novel sRNA species, by suggesting a model sRNA candidate for future studies of 5' RNA capping, by exploring the regulation of untranslated RNAs in response to amino acid starvation, and by emphasizing the importance of recognizing and eliminating biases in established experimental approaches.