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

Abstract

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. KEYWORDS Drosophila; DEAD box helicase; Notch signaling; RNA-binding protein; RNA helicase N OTCH signaling is an evolutionary conserved process that mediates cell-cell communication, which ultimately regulates cell fate (Artavanis-Tsakonas et al. 1999). Notch signaling is critical for many developmental processes and aberrant notch signaling has been related to many human diseases including cancer (Gridley 2003). Notch encodes a trans-membrane receptor that comprises an extracellular domain (NECD) and an intracellular domain (NICD). Notch is expressed at the cell surface as a heterodimeric receptor that is a result of furin-dependent cleavage (S1) occurring in the trans-Golgi network. At the cell surface it physically interacts with the ligands that are expressed in the apposing cells. This interaction with the ligand facilitates a series of proteolytic cleavages, ultimately resulting in the release of NICD. Released NICD translocates into the nucleus, where it interacts with a DNA binding protein CSL (mammalian CBF1/Drosophila Suppressor of Hairless/C. elegans Lag-1) and activates downstream gene expression, by relieving the repressor complex that silences Notch target genes (ArtavanisTsakonas et al. 1983;Logeat et al. 1998;Struhl and Greenwald 1999;Brou et al. 2000;Kopan 2002;Lieber et al. 2002). Finetuning of Notch signaling is mediated by a vast num...

An interaction network of RNA-binding proteins involved inDrosophilaoogenesis

“…Consistent with the well-documented role of RNA helicases in oogenesis and fertility (87,(123)(124)(125)(126), we identified two putative DEAD-box helicases, CG10077 and Mahe (Maheshvara). While CG10077 is an ortholog of human DDX5, Mahe is an evolutionary conserved regulator of Notch signaling (127). We could recapitulate the interaction of Mahe with both Hrp48 and Glo, and CG10077 with Glo in our co-IP assay.…”

Regulation of Notch Signaling by the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex in Drosophila melanogaster

“…Dx binds to the ANK repeats of Notch ICD via its N-terminal Notch-binding domain (Matsuno et al 1995). Different genetic and biochemical analyses suggest that Drosophila Dx, similar to mammalian Dx homologs, acts as a context-dependent modulator of Notch signaling (Diederich et al 1994;Matsuno et al 1995Matsuno et al , 1998Kishi et al 2001;Mukherjee et al 2005;Mishra et al 2014;Surabhi et al 2015). In Drosophila, Dx alone can positively regulate Notch signaling (Matsuno et al 1995).…”

“…Later, it was revealed that Shurb, a component of the ESCRT-III (endosomal sorting complexes required for transport-III) complex, modulates the Dx-Krz synergy (Hori et al 2011). Recent studies have also shown that Dx together with TRAF6 (a direct interactor of Notch) or Mahe (an RNA-binding protein) can downregulate the Notch pathway (Mishra et al 2014;Surabhi et al 2015).…”

“…Hrp48 along with Dx attenuates Notch signaling in an Sxl-independent manner Hrp48 is an RNA-binding protein, which is well-studied for its multiple roles during post-transcriptional modification of Oscar and Gurken mRNAs during oocyte development in Drosophila (Goodrich et al 2004;Huynh et al 2004;Yano et al 2004;Geng and Macdonald 2006;Giorgi and Moore 2007;Kalifa et al 2009). In recent years, the emerging role of different RNA-binding proteins in the regulation of Notch signaling has been studied in depth (Hall et al 2004;Saj et al 2010;Suissa et al 2010;Jung et al 2013;Surabhi et al 2015). Sxl binds to Notch mRNA and negatively regulates Notch signaling during wing disc development and oogenesis.…”

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

“…Drosophila stocks used for analysis were w 1118 (wild‐type), mahe EP1347 (hypomorphic allele for mahe ), and EP Δmahe d08059 (null mutant for mahe ). Embryo collection and immunostaining was done as described previously (Surabhi et al., ). Primary antibodies used were rabbit anti‐Mahe, 1:300; rat anti‐ELAV, 1:200; and mouse anti‐22c10, 1:100.…”