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.
Olga V. Artemyeva-Isman· Preprints.org· 0 citations
The mainstream approach to splicing therapy today involves synthetic antisense oligonucleotides. ASOs interfere with spliceosomal ribozyme assembly by blocking splice sites or influence protein regulators by blocking or adding their binding sites. ASOs are not compatible with human biology, which limits their efficacy and they come with a burden of chemical toxicity. Spliceosomal snRNAs adapted for a specific target can either enhance or suppress exon inclusion. Recombinant snRNAs with their pre-mRNA binding sites changed still assemble into functional snRNPs and integrate into active spliceosomes. Native to human cells, they are versatile for in vivo delivery and can be either encoded and expressed from rAAVs or delivered as RNA molecules by exosomes, benefitting from excellent biodistribution. Imported snRNAs are taken up by cytoplasmic maturation factors and transferred into the nucleus. Adapted snRNAs can be used to target different stage spliceosomes: U1 and U2 for early complexes, U2/U6 for precatalytic or U5 for catalytic complexes. U1 is the only spliceosomal snRNA thoroughly examined in pre-clinical studies (~100 mutations in 24 genes) and it is necessary to review the past 20 years of experience with U1 before moving on to other snRNAs. U1 is involved in the initial splice site selection and usually binds the 5’ss, but it also promotes 5’ss usage ‘at a distance’ if bound in the vicinity. Adapted U1 suppresses 3’ss usage, as likely does WT U1 if 3’ss CAG|GU protosplice site repeat is followed by a sequence resembling the start of the intron. However, historically the non-spliceosomal U7-OPT is often used as a scaffold molecule for exon-skipping ASOs. This chimeric U7 snRNA cannot join histone bodies, its WT destination. While both U1 and U7 show promising safety profiles in mice, WT U7 expression is 1000 times lower, than U1. U7 snRNA modulates master transcription regulators by binding histone-fold domain protein NF-Y; the effects of U7-OPT overexpression remain underexplored. U1, a splicing molecule, naturally works better for promoting exon inclusion, than U7, but years of experiments also revealed U1 and U7 limitations. New adaptations of the core spliceosomal snRNAs are necessary to overcome these limitations.
Olga V. Artemyeva-Isman· Preprints.org· 0 citations
U2, U6 and U5 are the components of spliceosomal ribozymes that descend from mobile self-splicing introns. The fragmentation of ancestral introns in eukaryogenesis led to the evolution of the ribozyme assembly in trans using common snRNAs and protein-chaperones. This stepwise process provides opportunities to target distinct stages of ribozyme folding. Reviewing successive spliceosomal complexes, we will discuss how to adapt them for therapy, both splicing modulation and permanent gene therapy. Adapting U2 and U6 snRNAs can overcome the limitations of U1 and U7, currently used to target early spliceosome complexes. U2 can select alternative 3’ss, such as VEGFA switch between pro- and anti-angiogenic isoforms. U6 and U2 can be used together in the pre-catalytic complex, taking advantage of the U6/U2 Helix II that can be modified to prevent intermixing with WT U6 and U2 molecules. Extending 5’ss and BP helices with adapted U6 and U2 can create specific spliceosome species for individual introns. U5 snRNA, responsible for exon recognition, is the last to pair with pre-mRNA at pre-catalytic stage, but in reverse splicing, the exon recognition loop initiates intron insertion. Completed forward splicing produces an RNA-protein complex of an intron lariat paired with U6 and U2, the active ribozyme centre and U5 snRNA associated by protein interactions – the Intron Lariat Spliceosome. ILS is homologous to the mobile Group IIA intron particle capable of invading genomic loci by reverse splicing. Group II introns are used for microbial genome engineering. Can human ILS be adapted for specific genome insertions? Group IIA introns possess a second exon-recognition loop, which adds 6bp to the interaction with the 5’exon. Engineering an additional exon-recognition loop for spliceosomal U5 snRNA is the way to bring back reverse splicing. The field of genome engineering is dominated by CRISPR/Cas derivatives, so why do we need to develop reverse splicing? It is increasingly apparent that the evolutionary fate of RNPs defines their therapeutic utility: CRISPR/Cas are prokaryotic RNPs. Targeting human RNPs is the best solution, as the therapeutic success of RNAi using RISC complex indicates. The spliceosome is a human RNP worth exploring for splicing and gene therapy.
Olga V. Artemyeva-Isman· Preprints.org· 0 citations
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