Abstract:
Fas associating Factors (Faf1/Casp and Faf2) are members of the UBX domain
containing proteasomal adaptors family of proteins. The primary sequence analysis
suggests conservation of structure and function between flies and humans. Casp has
been previously reported to be involved in Drosophila innate immunity and also has
been characterized as a maternal effect gene in development. The physiological
function of Faf2 is not well characterized in Drosophila.
In this study, we attempt to characterize the role of the different functional
domains of Casp by generating Casp transgenic animals with targeted domain
deletions. The deletions of interest were the ΔUBX, and a combined deletion
ΔUASΔUBX. Our first attempt used CRISPR/Cas9 based genome editing, which did
not succeed. As an alternative, we were successful in generating a number of
overexpression transgenic animals, pUASP-ΔDomain flies, which we plan to express
in a Caspnull animal using the UAS Gal4 system. We are also redoing the CRISPR
screen using a modified design. Additionally, a loss of function analysis using reverse
genetics on the Casp homolog Faf2 suggest that Faf2 is a paternal effect gene. Zygotic
copy of Faf2 is not sufficient to rescue the paternal phenotype. Faf2 knockdown does
not cause sterility of the male germline as the embryos fathered by Faf2 knockdown
males proceed through nuclear divisions. Paternal germline specific Faf2 knockdown
results in embryonic developmental arrest at early stages of gastrulation.
Given that Faf2 is not present in the Drosophila sperm and that Faf2 influences
protein dynamics in the cell, we hypothesize that Faf2 functions in the observed
paternal effect phenotype by altering the chromatin architecture of paternal
chromosomes.
