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

Design Of Lunar-Gravity-Assisted Escape Trajectories

AbstractLunar gravity assist is a means to boost the energy and C3 of an escape trajectory. Trajectories with two lunar gravity assists are considered and analyzed. Two approaches are applied and tested for the design of missions aimed at Near-Earth asteroids. In the first method, indirect optimization of the heliocentric leg is combined to an approximate analytical treatment of the geocentric phase for short escape trajectories. In the second method, the results of pre-computed maps of escape C3 are employed for the design of longer Sun-perturbed escape sequences combined with direct optimization of the heliocentric leg. Features are compared and suggestions about a combined use of the approaches are presented. The techniques are efficiently applied to the design of a mission to a near-Earth asteroid

Missions for Asteroid Insertion into Earth-Mars Cycler

This paper analyzes the feasibility of insertion of a 1000-metric ton asteroid into an Earth-Mars cycler. An electric propulsion space tug comparable to the one selected for the now-canceled Asteroid Robotic Redirect Mission is considered. An indirect optimization method is used to compute the low-thrust trajectories. Resonant and non-resonant trajectories that allow for multiple Earth encounters are explored to define the trajectory structure in order to limit the propellant consumption to feasible values. Two examples for the insertion of asteroid 2010 UY7 into Earth-Mars S1L1 cyclers are presented

Optimal Design of Electrically Fed Hybrid Mars Ascent Vehicle

The optimal design of the propulsion system for a potential Mars Ascent Vehicle is analyzed, in the context of the Mars Sample Return Mission. The Mars Ascent Vehicle has to perform an initial ascent phase from the surface and then circularize into a 170 km orbit. A two-stage launcher is taken into account: the same hybrid rocket engine is considered for both stages in order to limit the development costs. A cluster of two, three or four engines is employed in the first stage, whereas a single engine is always used in the second stage. Concerning the feeding system, three alternatives are taken into consideration, namely a blow down, a regulated and an electric turbo-pump feed system. The latter employs an electric motor to drive the oxidizer turbopump, whereas the power is supplied to the motor by lithium batteries. All the design options resulted in viable Mars Ascent Vehicle configurations (payloads are in the range of 70–100 kg), making the hybrid alternative worth considering for the sample return mission. The use of an electric turbo-pump feed system determines the highest vehicle performance with an estimated 10–25% payload gain with respect to gas-pressure feed systems

Radial Turbine Thermo-Mechanical Stress Optimization by Multidisciplinary Discrete Adjoint Method

This paper addresses the problem of the design optimization of turbomachinery components under thermo-mechanical constraints, with focus on a radial turbine impeller for turbocharger applications. Typically, turbine components operate at high temperatures and are exposed to important thermal gradients, leading to thermal stresses. Dealing with such structural requirements necessitates the optimization algorithms to operate a coupling between fluid and structural solvers that is computationally intensive. To reduce the cost during the optimization, a novel multiphysics gradient-based approach is developed in this work, integrating a Conjugate Heat Transfer procedure by means of a partitioned coupling technique. The discrete adjoint framework allows for the ecient computation of the gradients of the thermo-mechanical constraint with respect to a large number of design variables. The contribution of the thermal strains to the sensitivities of the cost function extends the multidisciplinary outlook of the optimization and the accuracy of its predictions, with the aim of reducing the empirical safety factors applied to the design process. Finally, a turbine impeller is analyzed in a demanding operative condition and the gradient information results in a perturbation of the grid coordinates, reducing the stresses at the rotor back-plate, as a demonstration of the suitability of the presented method

GTOC X: Solution Approach of Team Sapienza-PoliTo

This paper summarizes the solution approach and the numerical methods developed by the joint team Sapienza University of Rome and Politecnico di Torino (Team Sapienza-PoliTo) in the context of the 10th Global Trajectory Optimization Competition. The proposed method is based on a preliminary partition of the galaxy into several small zones of interest, where partial settlement trees are developed, in order to match a (theoretical) optimal star distribution. A multi-settler stochastic Beam Best-First Search, that exploits a guided multi-star multi-vessel transition logic, is proposed for solving a coverage problem, where the number of stars to capture and their distribution within a zone is assigned. The star-to-star transfers were then optimized through an indirect procedure. A number of refinements, involving settle time re-optimization, explosion, and pruning, were also investigated. The submitted 1013-star solution, as well as an enhanced 1200-point rework, are presented