Alternative splicing of pre-messenger RNA increases genetic diversity and recent studies estimate that most human multi-exon genes are alternatively spliced. If this process is not highly regulated and accurate, it leads to mis-splicing events which may result in proteins with altered function. A growing body of work has implicated mis-splicing events as contributing to a wide range of diseases including cancer, neurodegenerative diseases, and muscular dystrophies. Understanding the mechanisms that cause aberrant splicing events and how this leads to disease is vital for designing effective therapeutic strategies. This review focuses on advances in therapies targeting splicing and highlights the animal models developed to recapitulate disease phenotypes as a model for testing these therapies.

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNAPro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNAPro is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In the present study, experimental and computational approaches were used to test the hypothesis that INS deletion alters the internal protein dynamics leading to reduce catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199–206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled-dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Taken together, the present study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Aminoacyl-tRNA synthetases are multi-domain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In the present study, we investigate the molecular mechanism of site-to-site communication in Escherichia coli prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved/co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis was performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue-residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics suggesting that any perturbation along these pathways disrupts the native residue-residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway, as well as cross-talk between pathways are important to coordinate functions of different domains of Ec ProRS for efficient catalysis.

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain -editing proteins. ProXp-ala is a ubiquitous-editing enzyme that edits Ala-tRNA, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNA and mischarged Ala-tRNA is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNA acceptor stem mimic, microhelix, or a nonhydrolyzable mischarged Ala-microhelix substrate analog identified residues important for binding and deacylation. Backbone N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a-editing domain to ensure acceptance of only mischarged Ala-tRNA, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.