Pre-mRNA Splicing: An Evolutionary Computational Journey from Ribozymes to Spliceosome

The intron\u2013exon organization of the genes is nowadays taken for granted and constitutes a fully established theory. DNA protein-coding sequences (exons) are not contiguous but rather separated by silent intervening fragments (introns), which must be removed in a process called pre-mRNA splicing. However, this fragmented composition of the eukaryotic genome has ancient origins. It appears that, during the initial stages of eukaryotic evolution, group II introns, i.e. self-splicing catalytic ribozymes, invaded the eukaryotic genome via the endosymbiosis of an alpha-proteobacterium in an archaeal host. At a later time, they split into the inert spliceosomal introns and the catalytically active small nuclear (sn)RNAs, which, together with additional splicing factors, gave rise to the eukaryotic spliceosome. This marked the transition from the autocatalytic splicing, mediated by ribozymes (RNA filaments endowed with an intrinsic catalytic activity) to splicing mediated by a protein-RNA machinery, the spliceosome. In the present thesis, the evolutionary relationship between group II introns and the spliceosome is retraced from a computational perspective by means of classical molecular dynamics simulations (MD), quantum mechanics calculations (QM) and combined quantum-classical simulations (QM/MM). The splicing process of these two different \u2013 but mechanistically related \u2013 large and sophisticated biomolecules is investigated with the aim of deciphering the reactivity and the structural properties from a computational point of view, with a focus on the role played by the Mg2+ ions as splicing cofactors. In Chapter 2, the importance of Mg2+ ions in the RNA biology is introduced. Not only they participate to the catalysis, but also represent essential structural and functional elements for RNA filaments. Moreover, the structural and molecular biology of group II intron ribozymes and the spliceosome machinery are widely discussed with a focus on their evolutionary links. Chapter 3 consists of a brief review of all the computational techniques employed in this thesis, from classical MD to QM and QM/MM simulations and enhanced sampling methods aimed at reconstructing the free energy of a process. Chapter 4 is entirely dedicated to the splicing mechanism promoted by group II intron ribozymes, representing the starting point of the evolutionary journey. In this chapter, a QM/MM study of the molecular mechanism of group II introns first-step hydrolytic splicing is presented, unveiling an RNA-adapted Steitz and Steitz\u2019s two-Mg2+-ion dissociative catalysis which differs from the one observed in protein enzymes. Chapter 5 is focused on Mg2+ ions, which are the natural cofactors of splicing, both in group II introns and in the spliceosome. Mg2+/RNA interplay is here addressed using a group II intron as a prototype of a large RNA molecule binding Mg2+. The performances of five different force fields currently used to describe Mg2+ in MD simulations are benchmarked, showing strengths and drawbacks. Moreover, the non-trivial electronic effects induced by Mg2+ on its ligands, such as charge transfer and polarization, are also characterized using 16 recurrent binding motifs. Overall, the study offers some guidelines on Mg2+ force fields for users and developers. Chapter 6 represents the final stop of the evolutionary journey. Here, an exquisite cryo-EM model of the ILS spliceosomal complex solved at 3.6 \uc5 resolution is used for a long-time scale MD study. This provides precious insights on the main proteins and snRNAs involved in the pre-mRNA splicing in eukaryotes as well as on the catalytic site. Unprecedentedly, the structural and dynamical properties of the spliceosome machinery are investigated at the atomistic level, with a particular emphasis on protein/RNA interplay through the characterization of their principal motions, among which the intron lariat/U2 snRNA helix unwinding

Molecular simulations and modeling of pharmaceutically relevant RNA-processing enzymes

The two-metal-ion architecture is a structural feature found in a variety of RNA processing metalloenzymes or ribozymes (RNA-based enzymes), which control the biogenesis and the metabolism of vital RNAs, including non-coding RNAs (ncRNAs). Notably, such ncRNAs are emerging as key players for the regulation of cellular homeostasis, and their altered expression has been often linked to the development of severe human pathologies, from cancer to mental disorders. Accordingly, understanding the biological processing of ncRNAs is foundational for the development of novel therapeutic strategies and tools. Here, we use state-of the-art molecular simulations, complemented with X-ray crystallography and biochemical experiments, to characterize the RNA processing cycle as catalyzed by two two-metal-ion enzymes: the group II intron ribozymes and the RNase H1. We show that multiple and diverse cations are strategically recruited at and timely released from the enzymes’ active site during catalysis. Such a controlled cations’ trafficking leads to the recursive formation and disruption of an extended two-metal ion architecture that is functional for RNA-hydrolysis – from substrate recruitment to product release. Importantly, we found that these cations’ binding sites are conserved among other RNA-processing machineries, including the human spliceosome and CRISPR-Cas systems, suggesting that an evolutionarily-converged catalytic strategy is adopted by these enzymes to process RNA molecules. Thus, our findings corroborate and sensibly extend the current knowledge of two-metal-ion enzymes, and support the design of novel drugs targeting RNA-processing metalloenzymes or ribozymes as well as the rational engineering of novel programmable gene-therapy tools

Computational Investigation of the Molecular Mechanisms Regulating Nucleic Acid Processing Metalloenzymes

Polymerases and nucleases are enzymes processing DNA and RNA. They are involved in crucial processes for cell life by performing the extension and the cleavage of nucleic acid chains during genome replication and maintenance. Additionally, both enzymes are often associated to several diseases, including cancer. In order to catalyze the reaction, most of them operate via the two-metal-ion mechanism. For this, despite showing relevant differences in structure, function and catalytic properties, they share common catalytic elements, which comprise the two catalytic ions and their first-shell acidic residues. Notably, recent studies of different metalloenzymes revealed the recurrent presence of additional elements surrounding the active site, thus suggesting an extended two-metal-ion-centered architecture. However, whether these elements have a catalytic function and what is their role is still unclear. In this work, using state-of-the-art computational techniques, second- and third-shell elements are showed to act in metallonucleases favoring the substrate positioning and leaving group release. In particular, in hExo1 a transient third metal ion is recruited and positioned near the two-metal-ion site by a structurally conserved acidic residue to assist the leaving group departure. Similarly, in hFEN1 second- and third-shell Arg/Lys residues operate the phosphate steering mechanism through (i) substrate recruitment, (ii) precise cleavage localization, and (iii) leaving group release. Importantly, structural comparisons of hExo1, hFEN1 and other metallonucleases suggest that similar catalytic mechanisms may be shared by other enzymes. Overall, the results obtained provide an extended vision on parallel strategies adopted by metalloenzymes, which employ divalent metal ion or positively charged residues to ensure efficient and specific catalysis. Furthermore, these outcomes may have implications for de novo enzyme engineering and/or drug design to modulate nucleic acid processing