ONCOMIRNA-BIOGENESIS · Biogenesis of Oncogenic MicroRNAs : from the structure of the microRNA processing complexes to the inhibition of the maturation of human oncogenes

Periodic Report Summary - ONCOMIRNA-BIOGENESIS (Biogenesis of Oncogenic MicroRNAs : from the structure of the microRNA processing complexes to the inhibition of the maturation of human oncogenes)

Summarise project objectives Micro-RNAs (miRNAs) are a recently identified class of small non-coding RNAs that are believed to regulate the expression of up to 30 % of human genes. The contribution of miRNAs in gene regulation is gaining considerable attention, particularly given the growing links between miRNA mis-function or expression and disease. Certain miRNAs have been demonstrated to display oncogenic-like phenotypic effects and their overexpression is heavily implicated in the development of cancers. MiRNAs mature via a two-step pathway involving two processing complexes: first Drosha-DCGR8 and second Dicer-PACT-TRBP. The two ribonucleases Drosha and Dicer are recruited by multi-domain RNA-binding accessory proteins that interact with specific, but currently unknown, structural features present in immature miRNAs. In this project I propose to perform an ambitious and comprehensive multidisciplinary analysis of biomolecular interaction networks involved in human miRNA biogenesis. An automated molecular biology approach will be employed to rapidly clone, express and screen soluble domains derived from each modular processing protein. I will use the latest fast NMR methods to rapidly assess protein and RNA folds, and protein and RNA interaction sites. These results will define the roles of each biogenesis protein, describe RNA recognition mechanisms and reveal the first atomic resolution picture of miRNA processing complexes. This structural evaluation will form the basis for the design and testing of inhibitors of miRNA biogenesis. Over-expression of oncogenic miRNAs correlates well with cancer. I propose to evaluate the possibility of using anti-sense oligonucleotides to block the regions of the immature miRNA recognized by the biogenesis machinery. The efficacy of these molecules will be explored in vivo with the view to developing innovative ways of probing miRNA function or potential new cancer therapies. Description of work performed to date - Bioinformatic identification of potential domains constructs from the multi-domain proteins involved in miRNA biogenesis - Initial and iterative design of constructs from miRNA biogenesis accessory proteins and Dicer - Automated cloning and expression testing combined with preliminary analysis by NMR spectroscopy - Biophysical and NMR spectroscopy analysis of soluble constructs of miRNA biogenesis processing machinery - Purification of insoluble protein constructs under denaturing conditions and initial refolding assays - Test-scale synthesis of oncogenic pre-miR155 - Optimisation of miRNA production in vitro - In vitro production of larger-scale quantities of pre-miR155 for structural studies Main results achieved to date - Optimisation of a protocol for expression testing and NMR analysis of domain constructs (Rasia et al., 2009) - >20 dsRBD constructs produced from DGCR8, PACT, TRBP and Dicer - Preliminary NMR assignments of dsRBDs from accessory protein TRBP - Analysis of inter- and intra-molecular interactions between dsRBD of TRBP and PACT - Production of mg quantities of RNA for NMR analysis - Development of isotope-labelling strategies for NMR-based analysis of biomolecular interfaces in higher molecular weight complexes (Gans et al., 2010) Expected final results - Structural analysis of miRNA biogenesis machinery - Backbone NMR assignments of each soluble dsRBD - Atomic resolution structural analysis of each dsRBD - Mapping of protein-protein interactions using NMR spectroscopy and other biophysical and biochemical techniques - Mapping of protein-RNA interactions using NMR spectroscopy and other biophysical and biochemical techniques - Elucidation of a high-resolution structural model that describes the critical biomolecular interfaces and interactions involved in miRNA biogenesis.

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