Wang, H.-K. | Search bioRxiv

bioRxiv2025

A topologically conserved unstructured region helps positioning the evolutionarily conserved Prp40 WW domains to promote non-canonical intron splicing

Newly transcribed introns are immediately identified by early splicing factors that recognize the intron 5 splice site (5SS) and branch site (BS). In the budding yeast, these critical splice-site sequences are generally constrained, whereas degeneracy is the rule in higher eukaryotes. Yet, [~]40% of the yeast introns do diverge, to a certain degree, from the canonical sequences. Exactly how these non-canonical introns are recognized and spliced remains unknown. Here we show that the conserved Prp40 WW domains promote non-canonical intron splicing by enhancing stable U1 snRNP and BBP recruitments. AlphaFold predicts a topologically conserved unstructured region between Prp40 WW and FF domains. Alignment of the AlphaFold Prp40 structure with published U1 snRNP structure positions WW domains adjacent to 5SS and Luc7, which is known to be critical for 5SS recognition. Indeed, deletion of this unstructured region negatively impacts on splicing of the non-canonical 5SS introns. Taken together, our results suggest that the conserved WW domains may have evolved to deal with the highly degenerate 5SS and BS sequences in higher eukaryotes, so as to accommodate increased splicing complexity. HighlightsO_LIThe evolutionarily conserved Prp40 WW domains promote splicing of introns harboring non-canonical 5 splice site or branch site. C_LIO_LIPrp40 WW domains enhance stable U1 snRNP and BBP recruitments to nascent transcripts containing non-canonical splice sites. C_LIO_LIA topologically conserved unstructured region between WW and FF domains helps to position Prp40 WW domains close to the 5 splice site. C_LIO_LIThe N-terminal WW domain sterically hinders conformational rearrangements required for efficient release of a BBP variant during spliceosome assembly. C_LIO_LIA reporter assay identified 13 non-canonical introns whose splicing, under various environmental conditions, depend on Prp40 WW domains. C_LI

biochemistry↗

bioRxiv2025

A systemically delivered AAV-CFTR gene therapy for cystic fibrosis

Cystic fibrosis (CF) is the most common monogenic lung disease and results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). There have been over 2000 variants identified in patients that result in loss of function of the CFTR protein leading to systemic disease and respiratory failure in adolescence. While some variants encode proteins with residual activity that can be corrected or potentiated by CFTR modulators, at least 10% of CF individuals cannot tolerate the modulators or have nonsense mutations which fail to make any protein. For all people with CF, a mutation agnostic gene replacement strategy could provide a cure for CF lung disease. Here, we propose using a systemic route of administration to deliver a functional CFTR minigene cargo with a lung tropic AAV capsid. This would serve to reach multiple organs, most importantly the lung epithelium, and would provide a functional CFTR transgene that could be expressed in any cell type with a ubiquitous promoter. To achieve this, we generated the smallest CFTR minigene tested in an AAV delivery to date. We demonstrate its expression and function following transfection in cell-based assays and restoration of function in primary CF airway cells after viral delivery. Furthermore, we identify an AAV capsid that can transduce alveolar and airway epithelium with systemic delivery in non-human primates. These data provide tools for delivering a functional CFTR minigene that fits within the packaging capacity of an AAV and demonstrate lung transduction with an AAV following systemic delivery in a large animal model. This strategy first and foremost can reach target airway cells by circumventing the strong mucosal barrier in CF airways but may also provide a method by which to restore CFTR function in additional CF affected organs.

genetics↗