The chapter discusses the structure and function of the spliceosome, a multimegaladon ribonucleoprotein (RNP) complex responsible for pre-mRNA splicing. The spliceosome consists of five snRNPs and numerous proteins, and its conformation and composition are highly dynamic, allowing for both accuracy and flexibility in the splicing process. The dynamic nature of the spliceosome, which includes both RNA and protein components, suggests that it is an RNP enzyme. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their subunits, while X-ray and NMR studies have provided high-resolution structures of spliceosomal proteins. The chapter also highlights the importance of RNA-RNA and RNA-protein interactions in aligning the reactive groups of the pre-mRNA for catalysis, and the role of proteins in facilitating these interactions. The dynamic nature of the spliceosome during splicing is further emphasized by the reversible nature of the chemical steps of pre-mRNA splicing, which is similar to group II self-splicing introns. The chapter outlines the assembly pathways of the U2-dependent spliceosome, including the formation of various intermediate complexes and the rearrangements of RNA-RNA and RNA-protein interactions. It also discusses the dynamic network of RNA-RNA interactions within the spliceosome, particularly the conformational changes in U2 and U6 snRNAs during splicing. The chapter further explores the role of proteins in facilitating structural rearrangements in the spliceosome, such as the DEAD/H-box proteins and DExH/D-box proteins, and the importance of posttranslational modifications in promoting critical RNP rearrangements essential for splicing. Finally, it provides insights into the structural organization of the spliceosomal snRNPs and non-snRNP splicing factors, including the 3D structures determined by electron microscopy.The chapter discusses the structure and function of the spliceosome, a multimegaladon ribonucleoprotein (RNP) complex responsible for pre-mRNA splicing. The spliceosome consists of five snRNPs and numerous proteins, and its conformation and composition are highly dynamic, allowing for both accuracy and flexibility in the splicing process. The dynamic nature of the spliceosome, which includes both RNA and protein components, suggests that it is an RNP enzyme. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their subunits, while X-ray and NMR studies have provided high-resolution structures of spliceosomal proteins. The chapter also highlights the importance of RNA-RNA and RNA-protein interactions in aligning the reactive groups of the pre-mRNA for catalysis, and the role of proteins in facilitating these interactions. The dynamic nature of the spliceosome during splicing is further emphasized by the reversible nature of the chemical steps of pre-mRNA splicing, which is similar to group II self-splicing introns. The chapter outlines the assembly pathways of the U2-dependent spliceosome, including the formation of various intermediate complexes and the rearrangements of RNA-RNA and RNA-protein interactions. It also discusses the dynamic network of RNA-RNA interactions within the spliceosome, particularly the conformational changes in U2 and U6 snRNAs during splicing. The chapter further explores the role of proteins in facilitating structural rearrangements in the spliceosome, such as the DEAD/H-box proteins and DExH/D-box proteins, and the importance of posttranslational modifications in promoting critical RNP rearrangements essential for splicing. Finally, it provides insights into the structural organization of the spliceosomal snRNPs and non-snRNP splicing factors, including the 3D structures determined by electron microscopy.