Global Analysis Of The Human Rna Rna Interactome PDF Download

Numerous characterized non-coding (nc)RNAs function via base-pairing with target RNAs to direct essential roles in RNA processing, modification, turnover and translation. Moreover, functionally important RNA-RNA interactions have been defined in precursor (pre)-mRNA and spliced mRNAs. These previous studies therefore suggested that many additional important RNA-RNA interactions remain to be discovered and characterized. A major challenge in the field of RNA biology is therefore to systematically identify and characterize functionally important RNA-RNA interactions. Computational methods have yielded limited success, and previously there was a lack of global-scale experimental approaches to address this challenge. Accordingly, I developed and applied LIGR-Seq, a new method that enables the global-scale mapping of RNA-RNA interactions. LIGR-Seq involves cross-linking of RNA duplexes in cells and, following cell lysis and partial digestion and ligation of interacting RNA, high-throughput sequencing and mapping of the RNA ligation products to define RNA-RNA interactions. I used LIGR-Seq to map known and new ncRNA-mRNA interactions in human cells. These data led to the discovery that specific snoRNAs function in the regulation of turnover of target mRNAs, in addition to their well-established canonical roles in RNA modifications. I next developed and applied an 'enhanced' (e)LIGR-Seq protocol that affords more sensitive detection of RNA-RNA interactions. eLIGR-Seq data revealed a previously unknown RNA structure in primary transcripts encoding the small ribosomal protein subunit RPS26 that functions in its negative auto-regulation. Characterization of this and a competing RNA structure revealed that RPS26 protein functions in negative feedback regulation by binding and stabilizing a structure in its pre-mRNA that favours the formation of a non-expressed, circular RNA variant, over the corresponding, expressed linear mRNA. This finding thus represents a new function for RNA structure and circular isoforms. It further has implications for understanding Diamond-Blackfan anemias associated with mutations in RPS26. In summary, my thesis research has contributed a valuable method that has enabled the discovery and characterization of RNA-RNA interactomes. The results from this work are providing new insight into the roles of the myriad of uncharacterized inter- and intramolecular RNA-RNA interactions that underlie critical biological functions.