The Developmentally Active and Stress-Inducible Noncoding Hsr[omega] Gene Is a Novel Regulator of Apoptosis in Drosophila
Mallik, Moushami, Lakhotia, Subhash C., Genetics
The large nucleus limited noncoding hsrω-n RNA of Drosophila melanogaster is known to associate with a variety of heterogeneous nuclear RNA-binding proteins (hnRNPs) and certain other RNA-binding proteins to assemble the nucleoplasmic omega speckles. In this article, we show that RNAi-mediated depletion of this noncoding RNA dominantly suppresses apoptosis, in eye and other imaginal discs, triggered by induced expression of Rpr, Grim, or caspases (initiator as well as effector), all of which are key regulators/effectors of the canonical caspase-mediated cell death pathway. We also show, for the first time, a genetic interaction between the noncoding hsrω transcripts and the c-Jun N-terminal kinase (JNK) signaling pathway since downregulation of hsrω transcripts suppressed JNK activation. In addition, hsrω-RNAi also augmented the levels of Drosophila Inhibitor of Apoptosis Protein 1 (DIAP1) when apoptosis was activated. Suppression of induced cell death following depletion of hsrω transcripts was abrogated when the DIAP1-RNAi transgene was coexpressed. Our results suggest that the hsrω transcripts regulate cellular levels of DIAP1 via the hnRNP Hrb57A, which physically interacts with DIAP1, and any alteration in levels of the hsrω transcripts in eye disc cells enhances association between these two proteins. Our studies thus reveal a novel regulatory role of the hsrω noncoding RNA on the apoptotic cell death cascade through multiple paths. These observations add to the diversity of regulatory functions that the large noncoding RNAs carry out in the cells' life.
THE noncoding hsrω gene, located at the 93D4 cytogenetic region on the right arm of chromosome 3 of Drosophila melanogaster, produces several noncoding transcripts as the functional end products (reviewed in Lakhotia 2003). It is expressed in a developmentally regulated manner and is also inducible under conditions of stress like heat shock, amide treatment, and recovery from anoxia, etc. (Mutsuddi and Lakhotia 1995; Lakhotia 2003). Of the multiple noncoding transcripts produced by this locus, the nucleus limited hsrω-n transcript colocalizes with a variety of heterogeneous nuclear RNA-binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles, which act as dynamic sinks to regulate the trafficking of hnRNPs and other related RNA-binding proteins (Lakhotia et al. 1999; Prasanth et al. 2000; Jolly and Lakhotia 2006). The hsrω transcripts play a crucial role in normal development and differentiation in the fly, since nullisomy for this gene leads to a high degree of embryonic lethality (Ray and Lakhotia 1998). All Drosophila species show a functional homolog of this gene (Lakhotia and Singh 1982; E. Mutt and S. C. Lakhotia, unpublished data). To understand physiological roles of the noncoding hsrω gene, we established EP and RNAi lines (Mallik and Lakhotia 2009) designed to, respectively, overexpress or deplete hsrω transcript levels using the UAS/GAL4 expression system (Brand and Perrimon 1993). Initial studies showed that RNAi-mediated eye-specific depletion of hsrω transcripts rescued the retinal damage seen in flies with two copies of the GMR-GAL4 transgene. Furthermore, other studies in our laboratory demonstrated that misexpression of the hsrω transcripts modulates poly(Q)-induced neurodegeneration in fly models (Sengupta and Lakhotia 2006; Mallik and Lakhotia 2009). Since eye degeneration in GMR-GAL4 homozygous individuals is due to an elevated incidence of apoptosis (Kramer and Staveley 2003) and since proteins with expanded poly(Q) also trigger apoptosis (Sanchez et al. 1999; Evert et al. 2000; Gunawardena et al. 2003), we undertook the present study to examine if the hsrω transcripts play a role in the cell death pathway(s).
Apoptosis is a highly conserved and the most common form of programmed cell death (PCD), which is essential for normal development of multicellular organisms. Apoptosis requires activation of a conserved class of cysteine proteases or caspases to effect destruction of the cell (Lee and Baehrecke 2001). …