The modulation of RNA splicing by small molecules has emerged as a promising strategy for treating pathogenic infections, human genetic diseases, and cancer; however, the principles by which splicing modulation is achieved haven’t been clarified, not at the molecular level.

To see splicing modulation more clearly, scientists at the Istituto Italiano di Tecnologia (IIT) in Genoa and the European Molecular Biology Laboratory (EMBL) in Grenoble have zoomed in on the conserved active site of an RNA splicing machines, specifically, the self-splicing group II introns, the bacterial and organellar ancestors of the nuclear spliceosome. This work, which spanned enzymatic, computational, and crystallographic studies, has uncovered mechanistic details that could inform the rational design of splicing modulators through structure-based strategies.

The scientists presented their work in Nature Communications, in an article titled, “Targeting the conserved active site of splicing machines with specific and selective small molecule modulators.”

“Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how [the self-splicing group II introns] recognize small molecules through their conserved active site,” the article’s authors wrote. “These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression.

“Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.”

Cells rely heavily on the ability to finely control gene expression, a complex process by which the information contained in DNA is copied into RNA to eventually give rise to all the proteins and most of the regulatory molecules in the cell. This process includes splicing, a vital and ubiquitous reaction that ensures the correct maturation of transcribed genes in all forms of life.

Splicing, as the name suggests, involves “cut and paste” operations. These are necessary to create mature versions of RNA that can perform coding or noncoding functions.

“Studying the RNA splicing process is very complex due to the chemical reactions and the molecular actors involved, such as RNA, proteins, ions, and water molecules,” said Marco De Vivo, PhD, principal investigator of the Molecular Modeling and Drug Discovery Lab, associate director for computation at IIT in Genoa, and one of the paper’s senior authors. “Thanks to modern molecular simulation techniques, we have acquired a detailed understanding of what happens, and how to intervene to modulate splicing. Our study has already enabled us to synthesize new drug-like molecules capable of modulating splicing in a new, specific, and highly effective way.”

Indeed, IIT and EMBL researchers, with the support of EMBLEM—EMBL’s technology and knowledge transfer branch—and IIT’s patent office, have recently also deposited a patent that describes novel chemical compounds acting as splicing modulators. In the future, by further improving these compounds, it may become possible to regulate the production of specific proteins linked to defective or mutated genes.

The paper’s other senior author, Marco Marcia, PhD, group leader at EMBL Grenoble, remarked, “Visualizing splicing modulation at the near-atomic level is breathtaking. It allows us to control one of the most fundamental reactions in life. In the future, we will consolidate the successful integration of our biological experimental studies with the chemical and computational studies of our collaborators, aiming at an ambitious goal: to develop new drugs, such as antibacterials and antitumor agents.”

The researchers led by De Vivo and Marcia investigated the small molecule modulation of RNA splicing by integrating the EMBL’s and the Partnership for Structural Biology’s expertise in biochemistry, biophysics, and structural biology, and by using the automated MASSIF-1 beamline jointly operated by EMBL and the European Radiation Synchrotron Facility (ESRF) to obtain X-ray crystallographic structures of intron-ligand complexes. The researchers also leveraged IIT’s molecular dynamics simulation technology, which allowed for the study of the physico-chemical interactions of the molecules involved.

The study lays the groundwork for the future identification of potential drugs that act directly on genetic mutations or modifications which alter the process of gene expression, thereby targeting the onset of tumors or genetic diseases.

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