Studies on natural antisense RNAs and microRNAs
Sammanfattning: Regulatory RNAs are found in all kingdoms of life and involved in regulation of gene expression at various steps including RNA splicing, editing, stability, modification, export, translation and chromatin remodelling. A large number of regulatory RNAs have been described recently. Natural antisense RNAs are examples of regulatory RNAs that contain complementary sequences to other transcripts. They can be transcribed in cis from opposing DNA strands at the same locus or in trans from separate loci. Some natural antisense RNAs hybridise to their target RNA, forming doublestranded RNA. MicroRNAs are well known examples of trans acting natural antisense transcripts that partially hybridise to targets. To study regulatory RNAs, appropriate molecular tools are needed. Therefore, in this thesis we developed methods to detect physical RNA::RNA interactions, doublestranded RNA and microRNAs. We next applied these methods in a variety of biological systems. To study physical RNA::RNA interactions in growing cells, we considered the hok/sok toxinantitoxin plasmid stabilization locus of the R1 plasmid in Escherichia coli as a paradigm. We designed anti Sok peptide-peptide nucleic acid (peptide-PNA) oligomers that would inhibit hok mRNA::Sok-RNA interactions and release the hok toxin mRNA for translation. Results showed that anti Sok peptide-PNAs were bactericidal with phenotypes consistent with cell killing by Hok protein, supporting the model for hok mRNA::Sok-RNA interactions. We also observed that peptide-PNAs accumulate in cells and therefore are capable of efficient competition. Double-stranded RNAs can be created in cells by hybridisation of sense and antisense transcripts or by a fold-back RNA sequence. To study such complexes, we developed a two-step ligation method for enzymatic attachment of adaptors. The method can be applied to any double-stranded RNA fragment in its duplex form without a need for prior sequence or termini information. Using this method, we mapped a double-stranded region in the highly structured hok mRNA in Escherichia coli total RNA, providing evidence for its folded structure in cells. MicroRNAs regulate expression of their target mRNA through translation repression. Because of their short length, microRNAs are difficult to detect. We aimed to develop a method that could profile the expression of several microRNAs simultaneously by combining ligation of size-coded probes and PCR amplification. The new method is able to distinguish between microRNAs that differ by only one nucleotide and also detects only mature microRNAs. Using this method, we generated unique microRNA signatures for various cell and tissue samples.
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