Unraveling the Molecular Mechanism of Splicing through Molecular Dynamics Simulation

The genes architecture as made of intron and exons is now a widely accepted fact and a well-established hypothesis. Indeed, the exons regions of a DNA molecule that code for proteins are not a continuous unitary sequence, but silent intervening segments (introns) that must be eliminated in a process known as pre-mRNA splicing. However, the splicing process is far from being fully understood, as the subtle regulatory mechanisms underlying the generation of premature mRNA, hide a large number of unresolved biological questions. The main keeper of this enigma is represented by the spliceosome, a highly dynamic molecular machinery and the main actor of the splicing process. With recent technological advancement in structural biology, crystallographic techniques together with a remarkable and continuous improvement of computational tools, we are witnessing a breakthrough era that allows us to study and to understand at the atomic-scale key functional aspects of the working mechanisms of large biological macromolecules such as the spliceosome. In my Ph.D years, I have tackled mechanistic aspects of splicing process, trying to address to three main questions: (i) discovery of small molecules to target specific splicing factors for treatment of splicing-related diseases; (ii) unraveling the molecular mechanism at the basis of pre-mRNAs recognition and splicing fidelity; (iii) elucidating the structural and dynamical properties of the spliceosome and its allosteric regulatory networks. This have been achieved by the use of classical molecular dynamics simulations (MD), Metadynamics and Virtual screening simulations. In Chapter 2 I introduce you to the biological significance and conservation of the splicing and alternative splicing, how it is used in normal eukaryotic cells, as well as in cancer cells. The impact of deregulated splicing on a plethora of human diseases is also discussed as well. Finally, I will present the structural and molecular biology of the spliceosome machinery, also explaining how it can precisely process different pre-mRNA sequences. Chapter 3 reports a review of all the computational techniques that I have used in this thesis. Namely, a brief introduction to classical molecular dynamics simulations, virtual screening, enhanced sampling methods and network theory analysis is reported. Chapter 4 is entirely dedicated to identifying small molecules inhibitors for a particular kinase that is involved in pre-mRNA splicing and that contributes to the migration propensity of Triple Negative Breast Cancer, one of the most aggressive breast cancer types. Chapter 5 focuses on the early recognition mechanism of specific pre-mRNA sequences by the splicing cofactor U2AF2, that is also involved in the first steps of the spliceosome assembly. The recognition of these sequences represents one of the first pre-mRNA recognition events, and it underlies the alternative splicing of pre-mRNA, directing the spliceosome to generate one specific transcript rather than another, which result into distinct protein isoforms. The effect of cancer-associated mutations on this delicate recognition step is also investigated. Chapter 6 represents the first attempt at understanding the reshaping properties of the spliceosome and the allosteric signaling underlying it. In this chapter I report a MD simulations study based on the cryo-EM structure of a yeast spliceosome solved at near-atomic-level resolution. In particular, I have investigated the structural and dynamical properties of the spliceosome machinery, making use of network theory in order to trace the information exchange pathways at the basis of the characterized functional motions.