Spliceosomal Ribozyme Assembly

Abstract

The spliceosome became very complex in eukaryogenesis. The Group IIA intron progenitor was associated with a single protein, homologous to spliceosomal Prp8, but the LECA spliceosome included ~140 proteins. The acquisition of proteins was a neutral process, providing a pool of factors for the development of a coordinated assembly process, where proteins act as scaffold and chaperones, supporting RNA moieties. The RNA component of spliceosomal complexes is tiny. Structural studies offer us snapshots of protein re-arrangements remodelling the RNA. The spliceosome is commonly described as a ‘protein directed ribozyme’. What does this mean? Just how much control ribozymes can delegate to proteins? Spliceosomal ribozymes never lost their primary function of guiding catalysis by RNA base-pairing. To help with alternative splice site choices and to enforce precision, the spliceosome recruited another two small RNAs, U1 and U4, and still employs base-pairing. We discuss RNA structures central in spliceosomal and Group IIA intron ribozymes. Spliceosomal introns preserve protosplice site repeats CAG|GU at 5’ss and 3’ss that dictate a strict order of ribozyme folding. The demarcation of the 5’ss must involve the 3’ss in the downstream repeat. The distinct 5’-3’ss pair of spliceosomal introns serves to reconstruct the correct splice junction between the two repeats, preventing the intron ends from binding U5 snRNA Loop1. Although modern Group IIA introns never splice within repeats, structural and biochemical studies confirm 3’ss involvement at pre-catalytic stage. The exact configuration of the 5’-3’ss pair is different in Group IIA introns, but the parallel strands orientation is conserved. Our updated U5 model, that includes the 5’-3’ss pair, shows that the pre-mRNA strand flipping to achieve the local parallel orientation occurs after the short 3’exon duplex. This asymmetric 5’ and 3’exon binding with the recognition loop is shared with Group IIA introns. The exons are aligned for ligation on U5 Loop1, guided by Watson-Crick pairs as we have previously concluded based on positional dependencies at human splice sites. Our U5 model shows that exon duplexes of stacked pairs are demarcated from the 5’-3’ss pair by a gap, which is how the ribozyme structurally defines the cleavage sites.