All eukaryotic genomes contain, besides the coding information for amino acids in different proteins, a significant amount of noncoding sequences, which may or may not be transcribed. In general, the more evolved or biologically complex the organisms are, greater is the proportion of the noncoding component in their genomes. A resurgence of studies on the noncoding sequences in various genomes during the past several years makes it clear that the complex biological organization demands much more than a rich proteome. Although the more popularly known ncRNAs are the small microRNAs and other similar species, other types of larger ncRNAs with critical functions in regulating gene activity at various levels are being increasingly identified and characterized. A comprehensive understanding of the significance of noncoding DNA/RNA sequences in eukaryotic genomes is essential for understanding the origin and sustenance of complex biological organization of multicellular organisms.
For the past few decades our laboratory has been working on the noncoding hsrω gene in Drosophila. This developmentally active, stress-inducible gene situated at the 93D4 band on the right arm of chromosome 3, produces multiple transcripts with no protein coding potentiality (Mohler and Pardue 1982, Lakhotia and Mukherjee 1982, Garbe and Pardue 1986, Hovemann et al. 1986, Fini et al. 1989). It plays a crucial role in survival and normal development in the fly, since nullisomy for this gene leads to embryonic lethality. Among the major heat shock loci in Drosophila, 93D or hsrω forms one of the transcriptionally most active puffs after a temperature shock (Mukherjee and Lakhotia 1979) and is unique in its inducibility by a number of amides (Lakhotia and Mukherjee 1970, 1980, 1984, Tapadia and Lakhotia 1997). Of the several ncRNAs produced by this locus, the nucleus limited >10 Kb long hsrω -n transcripts form dynamic structures called omega speckles which harness a variety of heterogeneous nuclear RNA binding (hnRNPs) and other RNA processesing proteins and regulate their availability during normal as well as stress conditions (Lakhotia et al. 1999, Prasanth et al. 2000). The omega speckles are thus believed to function as storage sites for unengaged hnRNPs and depending upon the cellular needs, hnRNPs are dynamically released from and sequestered in the omega speckles (Lakhotia et al. 1999, Prasanth et al. 2000, Lakhotia 2003). Evolution of such RNA-based communication networks which perform hsrω -like functions and act as platforms for the association of specific proteomes with each of the varied nuclear sub-structures appear to be a fundamental prerequisite of all cells and organisms subjected to stress. Based on structural and functional similarities, it has been proposed that the large noncoding Sat III transcripts in human cells are functional analogues of the hsrω transcripts (Jolly and Lakhotia 2006).
To better understand physiological roles of the hsrω gene, both gain-of-function and loss-of-function genetic studies were employed to over-express and ablate this gene’s transcripts by using tissue-specific GAL4 drivers. In accordance with previous reports using hsrω deficient lines, the present study (Chapter 3) also showed that the hsrω -n transcripts are critical for survival since ubiquitous over-expression or RNAi-mediated ablation of hsrω transcript levels resulted in organismal lethality. Levels of these transcripts apparently regulate sex-specific responses in the fly with global reduction in levels of hsrω transcripts resulting in death of more female than male progeny and over-expression causing greater mortality of males. Absence of the large nuclear hsrω -n RNA leads to loss of omega speckles resulting in a diffuse nuclear distribution of the hnRNPs associated with these transcripts. On the other hand, EP-mediated augmentation of endogenous hsrω transcript levels enhanced sequestration of the hnRNPs in a manner reminiscent of hnRNP distribution during stress conditions. Furthermore, RNAi-mediated depletion of hsrω transcripts following a 40 min heat treatment at 370C was found to impede trafficking of hnRNPs from the 93D locus to transcriptionally active sites on the chromatin. Over-expression of these transcripts results in functional depletion of cellular pools of hnRNPs and other RNA processesing proteins known to associate with the hsrω RNA. Conversely, in the absence of this RNA and thus omega speckles, these proteins cannot be efficiently directed to their proper cellular targets (transcriptionally active chromatin sites). Either of these conditions result in dysregulation of downstream events. I have further discovered that the 93D locus produces a fourth noncoding transcript which is essentially identical to the nuclear hsrω-n RNA, except that it does not contain the 700 bp intron. Immunoprecipitation experiments revealed that this novel transcript is also a component of the omega speckles. However, functional characterization of this splice variant of hsrω n needs further studies.
During the course of my work I have established a new mechanism of how levels of the Drosophila Inhibitor of Apoptosis Protein 1 (DIAP1), and thus apoptosis, are regulated. Data presented in Chapter 4 demonstrate that depletion of this ncRNA dominantly suppressed apoptosis-induced by ectopic expression of the key regulators/effectors of caspase-mediated cell death in Drosophila by augmenting the levels of DIAP1. Further, suppression of induced cell death following depletion of hsrω transcripts was abrogated when the DIAP1-RNAi transgene was co-expressed. These results indicate 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. In addition, I also show, for the first time, a genetic interaction between the hsrω transcripts and the JNK signaling pathway since down regulation of hsrω transcripts suppressed JNK activation. Data presented here revealed a novel regulatory role of the hsrω ncRNA at multiple levels in the apoptotic cell death pathways.
In earlier studies, over-expression of the hsrω transcripts was shown to dominantly enhance polyQ-induced cytotoxicity in Drosophila (Sengupta and Lakhotia 2006). In the present study (Chapter 5), I show that down regulation of hsrω-n transcripts ameliorates pathogenesis in several Drosophila models of human polyQ neurodegenerative diseases. Loss of hsrω RNA not only suppressed the eye-specific degeneration mediated by GMR-GAL4 driven expression of the 127Q or MJDtr-Q78 or ataxin1 82Q or httex1p Q93 transgene, but also rescued premature death of flies expressing the expanded polyQ proteins pan-neuronally. I further demonstrate that the morphological and functional rescue of polyQ toxicity observed upon depletion of hsrω-n transcripts is associated with substantial reduction of polyQ protein aggregation without affecting transcription of the 127Q transgene. Reduction in hsrω transcript levels also abolished the induction of Hsp70 in the polyQ expressing cells. These results suggest that the hsrω transcripts have a role in early stages of polyQ aggregate formation. I have also investigated possible pathway/s through which the hsrω transcripts modulate polyQ-induced neurodegeneration in Drosophila. I show that down regulation of hsrω -n transcripts in a polyQ background suppressed the enhancing effects brought about by reduced availability of the hnRNP Hrb87F and of the transcriptional regulator, CREB Binding Protein (CBP). This study revealed, for the first time, that CBP and hnRNPs like Hrb87F and Hrb57A physically interact with each other and depletion of hsrω -n RNA enhances cellular levels of CBP possibly through its association with hnRNPs. Over-expression of hsrω and/or polyQ proteins disrupted the proteasomal activity. On the other hand, while hsrω-RNAi partially rescued eye damage following compromised proteasome activity, its ability to rescue polyQ toxicity was affected by sub-normal proteasomal activity. Thus, depletion of hsrω transcripts seems to rescue polyQ toxicity by simultaneously targeting several pathways which in turn suppress formation of polyQ aggregates and transcriptional dysregulation in polyQ expressing cells to prevent neurodegeneration and preserve neuronal function.
Identification and elucidation of diverse novel roles played by ncRNAs such as the hsrω transcripts thus provide a new paradigm for understanding the complex regulation of nuclear RNA processing activities. The present study clearly demonstrates that such large ncRNA species contain additional information or functions which are as important as the protein coding genes. It is intriguing that these transcripts influence intricate networks of gene expression that result in precise organization of living organisms, not only in the nucleus but also in the cytoplasm. These transcripts apparently function as endogenous communication systems or hubs, which are central to the genetic control architecture not only within but also between cells, for coordination of many genes at different levels and thus ensure homeostasis. Such multiple networking interactions provide a basis for the context-dependent actions of the same molecule in different cells or in same cell under different conditions.