Design of miR-34a mimics shifting the targetome towards HNF4

microRNAs (miRNAs) regulate gene expression by inhibiting translation or inducing the degradation of messenger RNAs (mRNAs). miR-34a acts as a tumor suppressor, is predicted to have thousands of targets with more than hundreds of them are validated. Dysregulation of miR-34a has been implicated in various diseases.

In this thesis, we investigated the structural and functional aspects of miRNA mediated repression, exploring how the structure governs the function of miRNA. We developed a structure-based design approach to increase specificity of miR- 34a. We than used this to design a series of miR-34a mimics aiming at specifically trapping a subset of the targets, centered around hepatocyte nuclear factor 4 alpha (HNF4a), which has been reported to be dysregulated in several cancer types. We used dual-luciferase reporter assay to measure the downregulation efficiency and RNA:RNA binding by SHAPE (RABS) to characterize the structural features. We found that the mimic with an altered tail region exhibited increased down-regulation activity for HNF4a specifically, while having little or no effect on the other targets in the luciferase reporter system. We highlight the critical importance of tail region of miR-34a, where three cytosines are located, with two of them being adjacent. We evaluated the overall effects of the designed miRNA mimics on the transcriptome via RNA sequencing and demonstrated that the mimics were able to capture mRNA targets sharing similar structural features with HNF4a. By designing a mimic to stabilize tail binding of miR-34a:target interaction, we successfully shifted miR-34a targetome, thus creating a specific yet broad interaction, which could provide insights for the design of miRNA-based drugs to treat multigenetic diseases.

This thesis includes three projects. In Project I, we established a structural probing methodology RABS (RNA:RNA binding by SHAPE) to characterize the structural features of miRNA binding to its mRNA target. In project II, we investigated a specific miR-34a:HNF4a interaction and designed several miR-34a mimics to increase the specificity to HNF4a. In project III, we designed miRNA based small RNAs, which could shift miRNA targetome to a subset of target mRNAs. We showed the proof of concept for structure-activity relationship design of miRNA- based mimics to modulate target specificity, which may contribute to the concept of miRNA-based drug development.

List of scientific papers

I. Elnaz Banijamali, Lorenzo Baronti, Walter Becker, Joanna J Sajkowska-Kozielewicz, Ting Huang, Christina Palka, David Kosek, Lara Sweetapple, Juliane Müller, Michael D Stone, Emma R Andersson, Katja Petzold. RNA:RNA interaction in ternary complexes resolved by chemical probing. RNA. 2023 Mar;29(3):317-329 https://doi.org/10.1261/rna.079190.122

II. Ting Huang, David Kosek, Rubin Dasgupta, Joanna J Sajkowska- Kozielewicz, Emma R Andersson, Katja Petzold. Design of miR-34a mimics shifting the targetome towards HNF4x for treatment of cancer [Manuscript]

III. Ting Huang, Emma R Andersson, Katja Petzold. Design of small RNAs shifting the targetome of microRNA [Manuscript]