Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog (dElys) plays critical roles during Drosophila development. dElys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid. The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that dElys has conserved structural features, but contains a noncanonical AT-hook–like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by dElys during development.

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function.Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency.RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatiotemporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

Nuclear pores are the exclusive conduit to facilitate the nucleocytoplasmic transport in a precisely regulated manner. ELYS, a constituent protein of nuclear pores, initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores towards the end of mitosis. Using cellular, molecular and genetic tools, here, we report that ELYS orthologue (dElys) plays critical roles during Drosophila development. Through in silico analyses, we find all conserved structural features in dElys except for the presence of non-canonical AT-hook motif strongly binding with DNA. dElys localized to nuclear rim in interphase cells, but during mitosis, it was present on chromatin. RNAi mediated depletion of dElys leads to aberrant development and defects in the nuclear lamina and NPCs assembly at the cellular level. Furthermore, we demonstrate that in dElys depletion NF-B is activated and accumulates inside the nucleus which results in illtimed expression of critical molecules. dElys depletion sustains NF-B into the nucleus in post-embryonic stages. Prolonged NF-B inside nucleus induces apoptosis in response to hitherto unknown quality check mechanism and highlights on the underappreciated apoptotic paradigm of NF-B pathway.