Viet, J. | bioRxiv Science

bioRxiv2023

Eye lens organoids going simple: characterization of a new 3-dimensional organoid model for lens development and pathology

The ocular lens, along with the cornea, focuses light on the retina to generate sharp images. Opacification of the lens, or cataract, is the leading cause of blindness worldwide. Presently, the best approach for cataract treatment is to surgically remove the diseased lens and replace it with an artificial implant. Although effective, this is costly and can have post-surgical complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and anti-cataract drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined 3-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization at the tissue, cellular and transcriptomic levels. These 3D cultured lens organoids can be rapidly produced in large amounts. High-throughput RNA-sequencing (RNA-seq) on specific organoid regions isolated by laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to induction of opacities. Finally, knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology, and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataract.

cell biology↗

bioRxiv2022

EXONIZATION BY THE EMERGENCE OF A CLEAVAGE-POLYADENYLATION SITE

Exonization is the evolutionary process of recruitment of new exonic regions from previously intronic regions. It is a major contributor to the increased complexity of alternative splicing. Here, we explore exonization mediated by the emergence of a novel cleavage-polyadenylation site in an intron. In Xenopus laevis, the tpm1 gene, which encodes muscular tropomyosin, contains alternative terminal exons. In adult muscles and embryonic hearts, exon 9A is joined to the terminal exon 9B. In embryonic somites, it is joined to the exonic region 9, which is transcribed from the intron immediately downstream of exon 9A. Consequently, exon 9A is either an internal exon when ligated to exon 9B, or a part of a terminal exon along with region 9. We show here that region 9 is present only in amphibians and coelacanths. This suggests that it emerged in sarcopterygians and was lost in amniotes. We used antisense morpholino oligonucleotides to mask the regions of tpm1 pre-mRNA that potentially regulate the inclusion of exon 9A9. This revealed that the definition of exon 9A9 relies on a weak cleavage-polyadenylation site and an intronic enhancer, but is independent of the 3 splice site. We demonstrate that RNAs containing exon 9B are toxic in somites. This may have contributed to the evolutionary pressure that led to the exonization of region 9 in sarcopterygians. These findings reveal the emergence of a novel cleavage-polyadenylation site that avoids the accumulation of a toxic RNA as a novel mechanism for exonization-mediated diversification of terminal exons.

evolutionary biology↗