Maheshvara, a Conserved RNA Helicase, Regulates Notch Signaling in Drosophila melanogaster.

Gene expression is regulated at multiple steps after generation of primary RNA transcripts, including mRNA processing, stability, and transport, along with co- and post-transcriptional regulation. These processes are controlled via the involvement of a multitude of RNA binding proteins (RBPs). Innumerable human diseases have been associated with altered expression of RNA binding proteins. In this chapter we have focused on Maheshvara (mahe) which encodes a putative DEAD box RNA helicase protein in Drosophila. We have recently reported that mahe plays an important role in regulation of Notch signaling. Fine tuning of Notch signaling is required at multiple steps and it's misregulation leads to a variety of human diseases. Additionally, mutation in DDX59, a human homolog of mahe results in broad neurological phenotypes associated with orofaciodigital syndrome. Drosophila mahe mutants show abnormal peripheral and central nervous system development that resemble neuropathology of patients having mutation in DDX59 gene. This chapter will help in advancing the knowledge as to how mahe regulates Notch signaling and nervous system development.

A loss-of-function homozygous mutation in DDX59 implicates a conserved DEAD-box RNA helicase in nervous system development and function.

We report on a homozygous frameshift deletion in DDX59 (c.185del: p.Phe62fs*13) in a family presenting with orofaciodigital syndrome phenotype associated with a broad neurological involvement characterized by microcephaly, intellectual disability, epilepsy, and white matter signal abnormalities associated with cortical and subcortical ischemic events. DDX59 encodes a DEAD-box RNA helicase and its role in brain function and neurological diseases is unclear. We showed a reduction of mutant cDNA and perturbation of SHH signaling from patient-derived cell lines; furthermore, analysis of human brain gene expression provides evidence that DDX59 is enriched in oligodendrocytes and might act within pathways of leukoencephalopathies-associated genes. We also characterized the neuronal phenotype of the Drosophila model using mutant mahe, the homolog of human DDX59, and showed that mahe loss-of-function mutant embryos exhibit impaired development of peripheral and central nervous system. Taken together, our results support a conserved role of this DEAD-box RNA helicase in neurological function.

Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster.

A-kinase anchoring protein (AKAP) comprises a family of scaffold proteins, which decides the subcellular localisation of a combination of signalling molecules. Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. We have earlier reported that Spoon suppresses ribonuclear foci formed by trinucleotide repeat expanded transcripts associated with Spinocerebellar Ataxia 8 neurodegeneration in Drosophila. However, the role of Spoonbill in cellular signalling was unexplored. In this report, we have unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. We have shown here that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-ß activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, we found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. Hence, we propose that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson's Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.

Conservation of oocyte development in germline cysts from Drosophila to mouse.

Recent studies show that pre-follicular mouse oogenesis takes place in germline cysts, highly conserved groups of oogonial cells connected by intercellular bridges that develop as nurse cells as well as an oocyte. Long studied in Drosophila and insect gametogenesis, female germline cysts acquire cytoskeletal polarity and traffic centrosomes and organelles between nurse cells and the oocyte to form the Balbiani body, a conserved marker of polarity. Mouse oocyte development and nurse cell dumping are supported by dynamic, cell-specific programs of germline gene expression. High levels of perinatal germ cell death in this species primarily result from programmed nurse cell turnover after transfer rather than defective oocyte production. The striking evolutionary conservation of early oogenesis mechanisms between distant animal groups strongly suggests that gametogenesis and early embryonic development in vertebrates and invertebrates share even more in common than currently believed.

Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster.

Maheshvara (mahe), an RNA helicase that is widely conserved across taxa, regulates Notch signaling and neuronal development in Drosophila. In order to identify novel components regulated by mahe, transcriptome profiling of ectopic mahe was carried out and this revealed striking upregulation of JAK/STAT pathway components like upd1, upd2, upd3, and socs36E. Further, significant downregulation of the pathway components in mahe loss-of-function mutant as well as upon lowering the level of mahe by RNAi, supported and strengthened our transcriptome data. Parallelly, we observed that mahe, induced caspase-dependent apoptosis in photoreceptor neurons, and this phenotype was significantly modulated by JAK/STAT pathway components. RNA immunoprecipitation unveiled the presence of JAK/STAT tyrosine kinase hopscotch (hop) transcripts in the complex immunoprecipitated with Mahe, which ultimately resulted in stabilization and elevation of hop transcripts. Additionally, we also observed the surge in activity of downstream transcription factor Stat92E, which is indicative of activation of the JAK/STAT signaling, and this in turn led to apoptosis via upregulation of hid. Taken together, our data provide a novel regulation of JAK/STAT pathway by RNA helicase Maheshvara, which ultimately promotes apoptosis.

Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster.

Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling.