428A Poster - 05. Reproduction and gametogenesis
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Jillian Gomez ^1,2; Myles Hammond ^2,3; Stephen Kucera ²; Brian Oliver ¹; Leif Benner ^1,4

Affiliations:
1) National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD; 2) Department of Biology, University of Tampa, Tampa, FL; 3) Integrative Genetics and Genomics, University of California-Davis, Davis, CA; 4) Department of Biology, Johns Hopkins University, Baltimore, MD

Keywords:
b. oogenesis; g. alternative splicing

Sxl is the master sex-determination gene and acts via a sex-specific splicing mechanism. The locus has often been discussed as two simple steps in females: the production of an early protein involved in establishing autoregulatory splicing and the production of a late protein maintaining the female-specific splice. We mapped a female sterile allele, fs(1)K741, to the Sxl locus through deficiency mapping and duplication rescue. We used whole genome sequencing to determine fs(1)K741 to be a single point mutation (C>T) in the male-specific exon 3 of Sxl. CRISPR-Cas9 and a ssOligo donor directed to the point mutation in fs(1)K741 were employed to recreate the mutation in wild-type females. This point mutation failed to complement fs(1)K741, confirming it to be the cause of sterility and that fs(1)K741 is an allele of Sxl, thus called Sxl^K741-1. RT-PCR of Sxl^K741-1 ovaries showed both male and female-specific Sxl splicing products, while RT-PCR on Sxl^K741-1 carcasses showed only female-specific Sxl splicing. This indicates that aberrant splicing is restricted to the ovary. We wanted to determine how this mutation in the male exon was disrupting Sxl splicing. Homozygous Sxl^K741-1 females have a temperature-sensitive sterility and lethality effect. At 18°C, homozygous females are 28% viable and 80% fertile. At 29°C, homozygous females are 37% viable and completely sterile. Two duplications, Dp(1;3)DC489 and Dp(1;3)DC490, rescued viability of Sxl^K741-1 females. However, only Dp(1;3)DC490 rescued fertility. The difference between the two duplications is that Dp(1;3)DC490 contains the full Sxl reading frame while Dp(1;3)DC489 only contains the early Sxl reading frame, possibly indicating that the viability defect in Sxl^K741-1 is due to aberrant early protein function. We created loss-of-function frameshift mutations specific to the early, late, and all proteins denoted as, Sxl^Early, Sxl^Late, and Sxl^All, respectively. Sxl^All failed to complement viability of Sxl^K741-1 at both 18°C and 29°C. Sxl^Late failed to complement viability at 18°C, but was subviable and subfertile, at 29°C. Sxl^Early complemented viability and fertility of Sxl^K741-1 at both 18°C and 29°C. These results are somewhat at odds with the duplication results since the duplication only containing the early protein rescued viability. Still, it is clear from these new mutants that Sxl^K741-1 is not deficient in the establishment step of Sxl splicing, and the mutation is likely disrupting the maintenance of Sxl autoregulation. Although Sxl has previously been thought of and described as a simple step-wise autoregulatory cascade initiated by the early protein and maintained by the late protein, Sxl^K741-1 clarifies that the autoregulatory process of Sxl is much more complex. Understanding how this mutation in the male exon of Sxl leads to subviability and sterility will help researchers further understand the complex genetics of Sxl.