The process of pre-mRNA splicing is fundamental to gene regulation in eukaryotes. Specific RNA sequences called introns, which do not code for amino acids, are removed during pre-mRNA splicing, and flanking sequences are spliced together as exons forming mRNAs that are exported to the cytosol. While the spliceosome, a complex macromolecular machinery, helps form the catalytic core for splicing reaction, the transesterification reaction itself is catalyzed by the pre-mRNA molecule. The transesterification reaction as well as intermediates generated during pre-mRNA splicing are surprisingly similar to those generated during self-splicing group II introns that are generally present within lower organisms, including bacteria and yeast. In eukaryotes, multiple but different combinations of cis-elements and trans-acting factors are associated with the removal of different introns. Thus, each individual pre-mRNA is processed through a specific combination of sequence elements (cis-elements) and interacting proteins (trans-acting factors). Depending upon the relative expression of trans-acting factors, some sequences can be either recognized as exons or removed as introns, a process called alternative-splicing. ; This is a manuscript of an article published as Baralle, Francisco E., Ravindra N. Singh, and Stefan Stamm. "RNA structure and splicing regulation." Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1862, no. 11-12 (2019): 194448. DOI:10.1016/j.bbagrm.2019.194448. Copyright 2019 Elsevier B.V. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Posted with permission.