Heat shock RNA ω : Biological Overview | References
Gene name - Heat shock RNA ω
Synonyms - hsr-omega, hsromega
Cytological map position - 93D4-93D5
Function - non-coding RNA
Keywords - regulation of heat shock response, nucleus-limited hsrω-n transcripts (hsrω-RB and RG on Flybase) interact with several RNA processing proteins and organize the nucleoplasmic omega speckles compartmentalization, management of aggregation-prone hnRNPs, the architectural RNA hsrω affects sub-cellular localization of Drosophila FUS to drive neurodiseases
Symbol - lncRNA:Hsrω
FlyBase ID: FBgn0001234
Genetic map position - chr3R:21,295,979-21,317,730
Cellular location - nuclear and cytoplasmic
|Recent literature||Lo Piccolo, L., Mochizuki, H. and Nagai, Y. (2019). The lncRNA hsromega regulates arginine dimethylation of human FUS to cause its proteasomal degradation in Drosophila. J Cell Sci. PubMed ID: 31519807
The lncRNAs play structural and regulatory roles on RNA-binding proteins (RBPs). However, the mechanisms by which lncRNAs regulate the neurodegenerative-causative RBP like FUS remain poorly understood. This study shows that knockdown of the lncRNA hsromega causes a shift in the methylation status of human FUS from mono- (MMA) to di-methylated (DMA) arginine via upregulation of the argininemethyl transferases 5 (PRMT5). This novel regulatory role is critical to FUS toxicity since the PRMT5-dependent dimethylation of FUS is required for its proteasomal degradation and causes a reduction of high levels of FUS. Moreover, this study shows that an increase of FUS determines a decline of both PRMT 1 and 5 transcripts leading to an accumulation of neurotoxic MMA-FUS. Therefore, overexpression of either PRMT1 or PRMT5 is able to rescue the FUS toxicity. These results highlight a novel role of lncRNAs in post-translation modification (PTM) of FUS and suggest a causal relationship between lncRNAs and dysfunctional PRMTs in the pathogenesis of FUSopathies.
A delayed organismic lethality has been reported in Drosophila following heat shock when developmentally active and stress-inducible noncoding hsrω-n transcripts were down-regulated during heat shock through hs-GAL4-driven expression of the hsrω-RNAi transgene, despite the characteristic elevation of all heat shock proteins (Hsp), including Hsp70. This study shows that hsrω-RNAi transgene expression prior to heat shock singularly prevents accumulation of Hsp70 in all larval tissues without affecting transcriptional induction of hsp70 genes and stability of their transcripts. Absence of the stress-induced Hsp70 accumulation was not due to higher levels of Hsc70 in hsrω-RNAi transgene-expressing tissues. Inhibition of proteasomal activity during heat shock restored high levels of the induced Hsp70, suggesting very rapid degradation of the Hsp70 even during the stress when hsrω-RNAi transgene was expressed ahead of heat shock. Unexpectedly, while complete absence of hsrω transcripts in hsrω66 homozygotes (hsrω-null) did not prevent high accumulation of heat shock-induced Hsp70, hsrω-RNAi transgene expression in hsrω-null background blocked Hsp70 accumulation. Nonspecific RNAi transgene expression did not affect Hsp70 induction. These observations reveal that, under certain conditions, the stress-induced Hsp70 can be selectively and rapidly targeted for proteasomal degradation even during heat shock. In the present case, the selective degradation of Hsp70 does not appear to be due to down-regulation of the hsrω-n transcripts per se; rather, this may be an indirect effect of the expression of hsrω-RNAi transgene whose RNA products may titrate away some RNA-binding proteins which may also be essential for stability of the induced Hsp70 (Singh, 2016).
Heat shock response is one of the most conserved cellular cascades that effectively protect cells and the organism from adverse environmental conditions like physiologically high temperature, oxidative stress, cytotoxins, etc.. While the classical hallmark of the cell stress response has been the rapid induction of different families of heat shock or stress proteins, multiple long noncoding RNAs (lncRNAs) are now also known to be involved in the cell stress response (Lakhotia, 2012b; Place, 2014). The 93D/hsrω gene of Drosophila, with its multiple transcripts, is the earliest known lncRNA gene having significant roles in development and in cell stress response (Lakhotia, 1982; Lakhotia, 2011). The hsrω gene comprises a proximal region (∼2.6 kb), with two exons, E1 (~475 bp) and E2 (~750 bp), an intron (~700 bp), and a distal >5-kb region carrying short tandem repeats of 280 bp, unique to the locus (Lakhotia, 2012a). Until recently, it was believed to produce two primary transcripts, viz., hsrω-n1 (hsrω-RB) and hsrω-pre-c (hsrω-RC) from which the intron is spliced out to produce the hsrω-n2 (hsrω-RG) and hsrω-c (hsrω-RA) transcripts, respectively (Lakhotia 2011). Out of these four hsrω transcripts (two primary and two processed), the hsrω-c is cytoplasmic, while the other three are nuclear. Recent annotation at the Flybase indicates that the hsrω gene is longer than previously believed and produces additional transcripts. Little is known about the newly annotated hsrω transcripts although other studies confirm the presence of these new transcripts (hsrω-RD and hsrω-RF) and that the nearly 21 kb hsrω-RF transcript is heat shock inducible. A very small 23-bp long translatable ORF is present in the hsrω-c of Drosophila melanogaster, although its translation product has not yet been identified (Singh, 2016).
The nucleus-limited hsrω-n transcripts (hsrω-RB and RG on Flybase) interact with several RNA processing proteins and organize the nucleoplasmic omega speckles (Prasanth, 2000; Jolly, 2006; Onorati, 2011; Singh and Lakhotia, 2015). The omega speckles function as storage sites for several RNA processing proteins, which are dynamically released from or sequestered by the hsrω-n transcripts according to cellular needs (Lakhotia, 1999; Lakhotia, 2011; 2012; Jolly, 2006; Singh, 2015). The hsrω-null individuals are poorly viable and are thermosensitive (reviewed in Lakhotia, 1989; 2011). A recent study (Lakhotia, 2012a) showed that conditional down- or up-regulation of the hsrω nuclear transcripts through hs-GAL4-driven activation of UAS-hsrω-RNAi transgene (Mallik, 2009a) or of EP alleles of hsrω, respectively, during heat shock results in delayed organismic lethality in spite of the characteristic elevation in cellular levels of the different HSPs, including the Hsp70. The delayed organismic lethality in these genotypes during recovery was correlated with the absence of omega speckles and a slow and incomplete restoration of the hnRNPs on developmentally active gene loci during recovery after heat shock (Lakhotia, 2012a). It is known (Mallik, 2011) that global activation of UAS-hsrω-RNAi transgene by Act-GAL4 driver also disrupts the omega speckles. Therefore, it was of interest to see how the disruption of omega speckles in unstressed cells through down- or up-regulation of hsrω transcripts affects their stress response. Accordingly, the present study has examined heat shock response in tissues where hsrω transcripts were down- or up-regulated sometime before the cells were exposed to heat shock. It was found that expression of UAS-hsrω-RNAi transgene in unstressed cells severely affected the cellular levels of Hsp70 during heat shock as well as during subsequent recovery. Interestingly, expression of the UAS-hsrω-RNAi transgene did not affect heat shock-induced transcription, transport, and stability of the hsp70 messenger RNAs (mRNAs) but enhanced rapid degradation of the synthesized Hsp70 through proteasomal pathway even when the cells were under stress (Singh, 2016).
The noncoding hsrω gene of D. melanogaster is developmentally expressed in almost all cell types and is one of the most highly induced genes following heat shock (Lakhotia, 2011). The nuclear transcripts of hsrω gene, hsrω-n1, and hsrω-n2 are essential for organization of the omega speckles which are believed to regulate the availability of various hnRNPs and certain other RNA-binding proteins (Prasanth, 2000; Mallik, 2011; Lakhotia, 2011; Singh, 2015). In an earlier study (Lakhotia, 2012a), it was seen that when the UAS-hsrω-RNAi transgene or an EP allele of hsrω (EP93D or EP3037) was activated during heat shock using the hs-GAL4 driver, the major heat shock genes and stress proteins like Hsp70 and Hsp83 were characteristically induced, yet all the individuals exhibited delayed death during recovery. This delayed death was correlated with the delayed restoration of RNA Pol II, HP1, and hnRNPs to the developmentally active gene loci when UAS-hsrω-RNAi transgene or an EP allele of hsrω was expressed during heat shock (Lakhotia, 2012b; Singh, 2016 and references therein).
The present study used Act-GAL4 or Sgs-GAL4 or GMR-GAL4 drivers, instead of the hs-GAL4 driver, to alter the levels of hsrω transcripts much before the tissues experienced heat shock. In these cases also, as in earlier study (Lakhotia, 2012a), a delayed restoration of hnRNPs, etc. to their normal chromosomal sites was seen during recovery from heat shock, which may be the major factor for the observed delayed death. The most striking effect of expression of the hsrω-RNAi transgene, but not of the EP3037 allele, prior to heat shock is the near complete absence of Hsp70 in heat-shocked cells. The former would down-regulate levels of hsrω-n transcripts while the latter would result in their elevated levels (Mallik, 2009b). The present results show that cells with down-regulated levels of hsrω transcripts fail to elevate the levels of Hsp70 when heat shocked, although Hsp83 showed the expected increase (Singh, 2016).
Normally, the stress-inducible Hsp70 shows the most robust increase in stressed cells and continues to remain so for a few hours even after the stress condition is withdrawn. Only in certain WT tissues, like the MT and midgut polytene cells, this protein's inducibility by heat shock follows a different pattern (Lakhotia, 1996; Lakhotia, 2002; Lakhotia, 2002; Roberts, 1999). However, the Hsp70 accumulation after heat shock failed to follow the pattern characteristic of even these tissues when the UAS-hsrω-RNAi transgene was expressed prior to heat shock (Singh, 2016).
The continued puffing at the Hsp70-encoding 87A and 87C sites even after 4-h recovery from heat shock in Act-GAL4>hsrω-RNAi-expressing salivary gland (SG) also appears to be a consequence of the greatly reduced levels of Hsp70 in these cells since an optimal level of Hsp70 is required to autoregulate the stress-induced heat shock genes, including the hsp70 gene copies. Several earlier studies showed that a subnormal induction of the 93D puff harboring the hsrω gene during heat shock affects puffing at the 87A and 87C sites in a condition-specific manner (reviewed in Lakhotia, 1989; Lakhotia, 2011). In the present study also, it was noted that the 87A puff was generally smaller than the 87C puff in heat-shocked SGs that were already expressing the UAS-hsrω-RNAi transgene. However, the unequal puffing of the 87A and 87C puffs is unlikely to be responsible for the drastically reduced levels of Hsp70 in these cells. Observations on in situ localization of active RNA Pol II and hsp70 transcripts on the heat shock-induced 87A and 87C puffs and elsewhere show that heat shock-induced active transcription of the hsp70 genes and transport of hsp70 mRNAs from nucleus to cytoplasm are not affected by expression of UAS-hsrω-RNAi under the Act-GAL4 driver. Since levels of heat shock-induced hsp70 transcripts, as detected by RT-PCR, remained comparable and immunostaining for a P-body-specific GW182 protein did not reveal any significant difference in the distribution of P-bodies in different genotypes, it is unlikely that the hsp70 transcripts are prematurely degraded when the UAS-hsrω-RNAi transgene is expressed prior to heat shock under Act-GAL4 or Sgs3-GAL4 or GMR-GAL4 drivers. This is also supported by the observation that even 10 min after heat shock, these glands failed to show the characteristic high induction of Hsp70 (Singh, 2016).
Specific function of the 1.2 kb cytoplasmic hsrω-c transcript during development or in stress response is not known; however, its small translatable ORF has been suggested to have some role in translational activities in cells. Although earlier studies (Mallik, 2009b, Lakhotia, 2009) indicated that expression of the hsrω 280-bp repeat sequence present in the hsrω-RNAi transgene does not affect the levels of hsrω-c transcripts, it remains possible that the levels of hsrω-pre-c and, therefore, of the hsrω-c transcripts too may be affected due to the process of RNAi amplification, which in turn may affect translatability of the hsp70 transcripts. Therefore, this study also examined heat shock-induced accumulation of Hsp70 in cells co-expressing UAS-hsrω-RNAi and UAS-hsrω-pre-c transgenes. Since in this case, also Hsp70 levels remained as low as in only hsrω-RNAi transgene-expressing cells, it is believed that the potentially reduced levels of hsrω-pre-c and hsrω-c transcripts are unlikely to be a reason for the observed low levels of Hsp70 in the heat-shocked hsrω-RNAi-expressing cells. These studies also show that global expression of hsrω-RNAi transgene does not affect the basal levels of different Hsc70 proteins in unstressed cells. The 7.10.3 antibody detects cognate as well as the induced members of the Hsp70 family in Drosophila. In agreement with results obtained with the 7Fb ab, which detects only the stress-inducible Hsp70, the 7.10.3 ab signal in heat-shocked samples from hsrω-RNAi transgene-expressing larvae was much less intense than in WT. Thus, autoregulation of Hsp70 induction by enhanced levels of Hsc70s does not appear to be a reason for noninduction of Hsp70 in hsrω-RNAi background (Singh, 2016).
The current observations suggest that the absence or very little presence of Hsp70 in cells that have been expressing the UAS-hsrω-RNAi transgene before they were exposed to the thermal stress is due to specific degradation of Hsp70 by the proteasomal machinery since, when the proteasome inhibitor was present during heat shock, the Hsp70 accumulated in UAS-hsrω-RNAi transgene-expressing cells to the same extent as in other genotypes. Earlier studies have indeed shown that expression of UAS-hsrω-RNAi transgene enhances proteasomal activity (Mallik, 2010). It is intriguing that the improved proteasomal activity specifically removes the stress-induced Hsp70 but not any other HSP. It is known that Hsp70 follows a different pathway for degradation than other HSPs and, therefore, might be singularly targeted in hsrω-RNAi transgene-expressing cells during heat shock and recovery. It is intriguing that Hsp70 does not accumulate in stressed cells only when the UAS-hsrω-RNAi transgene is activated sometime before the heat shock (present results), but its accumulation remains unaffected when this transgene is activated along with heat shock (Singh, 2016).
The stress-induced Hsp70 is known to be rapidly removed by proteasomal degradation in WT cells recovering from the stress following its ubiquitination by the carboxy terminus of Hsp70-binding protein (CHIP). This protein plays a dual role in the heat shock response by ubiquitination of misfolded proteins and presenting them to Hsp70 during stress, and when misfolded proteins are no longer available during recovery, CHIP ubiquitinates Hsp70 for proteasomal degradation. It remains to be seen if CHIP is involved in the unusually rapid and premature degradation of Hsp70 or some other factors are at play in cells that express the UAS-hsrω-RNAi transgene prior to heat shock (Singh, 2016).
The findings that hsrω-null (hsrω66 homozygous) condition does not affect the heat shock-induced massive accumulation of Hsp70 but expression of UAS-hsrω-RNAi transgene in hsrω-null background affects Hsp70 levels after heat shock were completely unexpected. The first condition may suggest that a complete absence of hsrω gene since fertilization, as in hsrω66 homozygotes, but not subthreshold levels of hsrω transcripts, as in cells expressing the hsrω-RNAi transgene prior to heat shock, may affect the Hsp70 accumulation. However, absence of heat shock-induced Hsp70 protein when the hsrω-RNAi transgene is expressed in hsrω-null background is very perplexing since these cells do not have any target for the RNAi, as none of the hsrω transcripts is present. This raises the intriguing possibility that the observed effect may not be a consequence of down-regulation of the hsrω transcripts per se but may be a consequence of this transgene's expression itself. The hsrω-RNAi transgene construct includes a monomer of the 280-bp tandem repeats at the hsrω gene, taken from the pDRM30 clone together with ~80 bp of flanking vector sequence. The SympUAS vector would cause transcription of both strands of the 280 bp plus the flanking vector sequence when GAL4 is available. The 280-bp repeat sequence of the hsrω-n transcripts binds with several proteins (Lakhotia, 2011; Onorati, 2011). It remains possible that binding of some protein/s to the sense and/or antisense strands over-produced by this transgene construct may lead to functional depletion of that/those protein/s, which in turn may affect stability of the heat shock-induced Hsp70. It is known that excessive production of some RNAs or triplet repeat expansion in certain RNAs sequesters specific proteins resulting in dysregulation of their activities and thus proteinopathies. The current results with three other RNAi transgenic lines (Act-GAL4>Egfr-RNAi or Act-GAL4>Hsp60C-RNAi or Act-GAL4>Hsp60D-RNAi), each of which includes a long (>400 bp) RNAi construct, show that the absence of inducible Hsp70 in hsrω-RNAi transgene-expressing background is specific for expression of the hsrω-RNAi transgene. The protein-binding activities of each of the two strands produced by the SympUAS-hsrω-RNAi transgene construct when activated by GAL4 need to be examined. The dsRNA-dependent protein kinase, protein kinase R (PKR), has been implicated in determining the stability of hsp70 mRNA in mammalian cells such that PKR-null cells do not show significant accumulation of Hsp70 when exposed to cell stress. Although the results suggest that the heat-shocked-induced hsp70 transcripts are not destabilized by hsrω-RNAi transgene expression, it would be interesting to examine if the PKR homologues in Drosophila affect Hsp70 protein's stability (Singh, 2016).
The present observations appear to be the first to show selective instability of heat shock-induced Hsp70 so that it gets targeted to proteasomal degradation as soon as synthesized while the other induced heat shock proteins remain stable. This adds a new dimension to the complexity of regulation of the cell stress response. This study also brings out additional issues that may need to be considered when applying experimental RNAi approach. Normally, one expects the sense strand of the RNAi construct to get degraded. However, if it does not happen and if the RNA sequence can bind with some proteins, unexpected consequences may also follow (Singh, 2016).
The nucleus limited long-noncoding hsromega-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsromega locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) was used in unstressed and stressed Drosophila cells. Omega speckles display size-dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsromega gene site after stress. Instead of moving towards the nuclear periphery located hsromega locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsromega locus in non-particulate form. Continuing transcription of hsromega during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsromega gene site although their residence periods at chromosomes and hsromega locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsromega-n transcripts is presented (Singh, 2015).
Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions' alteration and loss was collectively named proteinopathies. This study shows that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the 'architectural RNA' (arcRNA) hsromega and with hsromega-associated hnRNPs. Additionally, it was found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. These results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization (Lo Piccolo, 2018).
To confirm these interactions, a co-immunoprecipitation assay was conducted using anti-TBPH antibody starting from wild-type (WT) fresh larval nuclear extracts. Squid and Hrb87F are enriched in TBPH pulled-down fractions (Lo Piccolo, 2018).
Several in vitro experiments through proteomic studies and co-immunoprecipitation assay in HEK293 cells showed that in human cells TDP-43 interacts with the Drosophila orthologs of Squid and Hrb87F hnRNPs. This study confirmed these results in vivo, showing that in Drosophila tissue TBPH also interacts with Squid and Hrb87F hnRNP (Lo Piccolo, 2018).
Considering the co-localization between TBPH and Squid/Hrb87F hnRNPs, it was asked whether TBPH and hsrω could physically interact as well. To answer this question, an immunofluorescence and fluorescence RNA in situ hybridization was conducted. A physical interaction was observed between TBPH and the architectural RNA (arcRNA) hsrω in Malpighian tubules (MT) in vivo (Lo Piccolo, 2018).
As hnRNPs are known to shuttle between nucleus and cytoplasm, Western blot analysis was performed using a previously described method to produce in a single experiment nuclear and cytoplasmic protein fractions (NF and CF). These experiments were performed using MT and brain cells (BCs). Omega speckles are present in all the larval and adult Drosophila cell-type tissues but cells from Malpighian tubules were used for their large nuclear size, which allow a better understanding of nuclear bodies' distribution, as well as the eventual hsrω-interacting protein subcellular localization. BCs were characterized, as TBPH is largely expressed and has fundamental roles in the brain (Lo Piccolo, 2018).
The localization of TBPH in BCs is predominantly localized in the nucleus, but in BCs there is also a fraction of TBPH protein in the cytoplasm (Lo Piccolo, 2018b).
To rule out that the physical association observed between TBPH and hsrω was due to fortuitous interactions occurring during nuclear extract preparation, a cross-linking RNA-immuno-precipitation (CLIP-RIP) biochemical assay was performed using the anti-TBPH antibody on fixed larval nuclear extracts from brain cells. The CLIP-RIP data confirmed the specific interaction between TBPH and hsrω in the nuclear extract from the brain cells (+3.05-fold), compared to Rox1 (+0.77-fold) and U4 (+1.1-fold), two other abundant nuclear non-coding RNAs (Lo Piccolo, 2018).
It was thus demonstrated that TBPH, as previously reported for Hrb87F and Squid, is able to bind the hsrω RNA in vitro. Furthermore, using a gel shift assay employing an hsrω-n repeat unit (280b) transcribed in vitro and a full-length recombinant TBPH, it was shown that TBPH effectively retards hsrω RNA gel mobility. Finally, as seen for Hrb87F and Squid hnRNPs, the addition of ISWI protein in the reaction is a strong modulator of the interaction between TBPH-hsrω, changing the gel shift delay (Lo Piccolo, 2018).
In conclusion, these experiments confirmed the interaction of TBPH with hsrω arcRNA, Squid and Hrb87F hnRNPs in the omega speckles context. These results strongly suggest that, like Hrb87F and Squid, TBPH is another hnRNP belonging to the omega speckles complexes. Moreover, as shown for Squid and Hrb87F hnRNPs, ISWI function is essential for the modulation of TBPH/hsrω interaction (Lo Piccolo, 2018).
The chromatin remodeler ISWI is essential for a correct organization of the nucleoplasmic omega speckles (Oranati, 2011). Indeed, the organization and distribution of omega speckles are profoundly altered in ISWI null mutants when compared to wild-type cells. Omega speckles lose their dot shape and assume a trail shape distribution in the nucleus, suggesting a severe defect in their maturation or organization. Squid and Hrb87F hnRNPs also form trail-like structures in the nucleus of ISWI null mutants, showing that not only the distribution of the hsrω arcRNA, but also that of omega speckle-associated hnRNPs is compromised (Lo Piccolo, 2018).
Therefore, the distribution of TBPH protein was analyzed in ISWI null mutants to check if loss of ISWI could influence TBPH organization in omega speckles NBs as for Hrb87F and Squid hnRNPs. Remarkably, it was found that compared to wild-type cells, loss of ISWI function changes TBPH distribution in the context of omega speckles, inducing a dramatic alteration of TBPH sub-cellular localization. While in WT MT TBPH immunoreactive spots are nucleus limited, in ISWI null mutants' MT cytoplasmic TBPH-positive spots and trails were detected. Of note, these cytoplasmic spots show to be organized in different shapes, as indicated by arrows and arrowheads (Lo Piccolo, 2018).
These data were confirmed in vitro by Western blot of nuclear and cytoplasmic fractions in WT versus ISWI null mutant MT and BCs. In detail, this study showed that in ISWI null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT NF (+2.37-fold). It was also observed a similar phenomenon in BCs where it was found that in ISWI null mutant TBPH disappears from the NF while its cytoplasmic fraction (CF) is not significantly changed compared to WT (Lo Piccolo, 2018).
Analyzing in detail the ventral ganglion of WT and ISWI null larvae, it was observed that the mean intensity of TBPH in motoneuron nuclei of ISWI null mutants is reduced compared to WT (Lo Piccolo, 2018).
Interestingly, loss of Squid in the Squid- null mutant also affects the cellular distribution of TBPH and causes its aberrant cytoplasmic localization in MT. While in WT MT TBPH immunoreactive spots are nucleus-limited, TBPH-positive cytoplasmic spots and trails were detected in MT of Squid null mutants. These data were confirmed in vitro by performing Western blots of nuclear and cytoplasmic fractions in WT versus Squid null mutant MT and BCs. In detail, it was shown that in Squid null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT nuclear fraction (Lo Piccolo, 2018).
The human orthologs of Squid and Hrb87F proteins interact with TDP-43 to function cooperatively in RNA metabolism regulation. This study addressed whether TBPH interacts with Hrb87F and Squid in Drosophila cells as well. Double-immunofluorescence was conducted for TBPH/Hrb87F and TBPH/Squid and it was found that TBPH co-localizes in Malpighian tubules with Squid and Hrb87F hnRNPs in vivo (Lo Piccolo, 2018).
A similar phenomenon was observed in BCs where it was found that in Squid null mutant TBPH disappear from the NF while its cytoplasmic fraction is quite similar compared to the WT (Lo Piccolo, 2018).
Unlike Squid, Hrb87F does not affect TBPH subcellular distribution. To explain this result, the existence of a hierarchical order was hypothesized in omega speckles assembling, and it was speculated that Squid together with ISWI could be master regulators in the formation of physiologically functional hnRNP-hsrω complexes. In this case it could be hypothesized that the loss of Squid protein forces TBPH protein to escape the nucleus as a consequence of incorrect interaction among all omega speckle-associated hnRNPs (Lo Piccolo, 2018).
All these data collectively suggest that, in the Drosophila cells, the disorganization of omega speckles' compartments caused by loss of ISWI's role lead to a redistribution of TBPH protein from the nucleus to the cytoplasm. This could be a very important observation, considering that intracellular deposition of aggregated and ubiquitinated proteins are a prominent cyto-pathological feature of most neurodegenerative disorders frequently correlated with neural cell death (Lo Piccolo, 2018).
To explain all the results presented, it is hypothesized that loss of ISWI's function may indirectly affect TBPH distribution as a consequence of incorrect interaction among the omega speckle-associated hnRNPs and hsrω arcRNA. Indeed, while Squid and Hrb87F in ISWI null mutants are disorganized in their structure, but remain in the nucleus, TBPH seems to be more affected and to escape from the nucleus to the cytoplasm (Lo Piccolo, 2018).
For instance, the data reinforce the role of the chromatin remodeler ISWI in the modulation of the cellular localization of aggregation-prone proteins and show that the correct nuclear compartmentalization of TBPH hnRNP is dependent on nuclear body maintenance regulated by the chromatin remodeler. Finally, the data are in line with the recent findings showing that TDP-43-dependent reduction of the chromatin remodeler Chd1's recruitment to chromatin affects the induction of several key stress genes necessary to protect from diseases like ALS and FTD (Frontotemporal Dementia) (Lo Piccolo, 2018).
FUS is an aggregation-prone hnRNP involved in transcriptional and post-transcriptional regulation that aberrantly forms immunoreactive inclusion bodies in a range of neurological diseases classified as FUS-proteinopathies. Although FUS has been extensively examined, the underlying molecular mechanisms of these diseases have not yet been elucidated in detail. Previous work has shown that RNAi of the lncRNA hsromega altered the expression and sub-cellular localization of Drosophila FUS in the central nervous system of the fly. In order to obtain a clearer understanding of the role of hsromega in FUS toxicity, this study drove the expression of human FUS in Drosophila eyes with and without a hsromega RNAi background. hFUS was largely soluble and also able to form aggregates. As such, hFUS was toxic, inducing an aberrant eye morphology with the loss of pigmentation. The co-expression of hsromega double-stranded RNA reduced hFUS transcript levels and induced the formation of cytoplasmic non-toxic hFUS-LAMP1-insoluble inclusions. The combination of these events caused the titration of hFUS molar excess and a removal of hFUS aggregates to rescue toxicity. These results revealed the presence of a lncRNA-dependent pathway involved in the management of aggregation-prone hnRNPs, suggesting that properly formed FUS inclusions are not toxic to cells (Lo Piccolo, 2017b).
FUS-proteinopathies are a group of genetically and clinically heterogeneous diseases that manifest in different manners depending on the region affected, such as motor neuron diseases (ALS-FUS) or various forms of dementia, including frontotemporal lobar degeneration with FUS pathology (FTLD-FUS), atypical FTLD with ubiquitin pathology (aFTLD-U), and other distinct forms of FTLD such as neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD)1. A hallmark of these conditions is immunoreactive FUS inclusion bodies. Several missense mutations in the FUS gene have been associated with the abnormal cytoplasmic localization of pathological FUS inclusions that are positive for p62 and ubiquitin in amyotrophic lateral sclerosis (ALS); however, FUS mutations have not been detected in most sporadic or familial cases of FTLD-FUS6 (Lo Piccolo, 2017b).
A large number of animal models have been employed to elucidate the underlying molecular mechanisms of FUS-proteinopathies, and Drosophila melanogaster has been utilized to discern some important aspects of their pathogenesis due to the conservation of FUS protein functions during evolution. All phenotypes caused by the removal of the Drosophila FUS orthologue Cabeza (dFUS), such as decreased adult viability, locomotor disabilities, and a short life span may be rescued by the heterologous expression of human FUS (hFUS). Knockdown models and the cell-type specific inactivation of dFUS have both contributed to understanding of the important role of FUS in neuronal development and also highlight how the loss of its nuclear function may induce the defects typically observed in some FUS-proteinopathies. Moreover, heterologous expression in the fly revealed that hFUS has the ability to translocate to mitochondria through the mediation of Hsp60 and induces apoptosis as a consequence of mitochondrial damage. This recent finding showed that in the absence of mutations, higher FUS levels also represent a critical event in FUS-proteinopathies. Consistent with this finding, sequencing of the 3'-untranslated region (3'-UTR) of FUS mRNA in ALS patients, with no mutations in the currently known ALS-associated genes, identified four mutations linked to a marked increase in the accumulation of FUS18, indicating that not only the nuclear-cytoplasmic imbalance of the mutant FUS, but also alterations in the physiological levels of the wild-type (wt) contribute to the pathogenesis of ALS (Lo Piccolo, 2017b).
Research over the past decade has revealed that FUS is a RNA-binding protein of the FUS, EWSR1, and TAF15 (FET) family, is 526 amino acids long, and is characterized by an N-terminal domain enriched in glutamine, glycine, serine, and tyrosine residues (QGSY), an RNA-recognition motif (RRM), multiple RGG-repeat regions involved in RNA binding, a C2/C2 zinc finger motif, and a highly conserved C-terminal region. FUS is intrinsically prone to aggregate and plays multiple nuclear and cytoplasmic steps in RNA processing, thereby regulating the spatiotemporal fate of mRNA, i.e. subcellular localization, translation, or degradation. FUS has been associated with general and more specialized factors that influence the initiation of transcription, splicing regulation, micro-RNA processing, and, more recently, the formation of circular RNAs. FUS may be co-transcriptionally deposited onto nascent mRNAs and drive RNA decay, which maintains this interaction during mRNA transport to the cytoplasm. FUS has the ability to control its own levels through a potent auto-regulatory mechanism, and, thus, the overexpression of FUS negatively affects its own expression post-transcriptionally. FUS preferentially binds to long transcripts, and previous studies highlighted its ability to specifically control a large number of long non-coding RNAs (lncRNAs) with important functions at different levels in the central nervous system (CNS). On the other hand, lncRNAs are dysregulated upon the depletion or unavailability of functional FUS in diverse models and diseases28, which raises questions about how FUS-proteinopathies affect lncRNA-based mechanisms (Lo Piccolo, 2017b).
Despite the large number of new findings, further studies are required in order to obtain a better understanding of the pathogenesis of FUS-proteinopathies. Although the progression of these diseases may be attributed to the propagation of FUS misfolding and aggregation, previous studies also reported that FUS has an inherent ability to form diverse and distinct insoluble partitions under physiological conditions. Hence, the mechanisms by which cells manage aggregation-prone proteins and how FUS inclusions correlate with diseases are not completely understood. Moreover, although an increasing number of studies have clearly revealed the important role of lncRNAs in many neurological diseases, it has not yet been established whether they also play a role in the pathogenesis of FUS-proteinopathies (Lo Piccolo, 2017b).
It was recently reported that the RNAi of the Drosophila lncRNA hsrω negatively controlled the abundance of the dFUS transcript and altered the dFUS nuclear-cytoplasmic balance. This was the first study to show the lncRNA-based control of FUS transcription and, until now, it represents the first evidence for the sub-cellular localization of FUS being dependent on lncRNAs (Lo Piccolo, 2017b).
With the main aim to understand the role of hsrω in FUS-proteinopathies, this study drove the expression of hFUS in Drosophila eyes with and without a hsrω RNAi background. When expressed in the posterior region of the imaginal disc, hFUS was distributed in two fractions: mainly soluble and also able to form relative soluble aggregates. As such, hFUS was toxic, inducing an aberrant eye morphology with the loss of pigmentation in the corresponding adult flies. The co-expression of hsrω double-stranded RNA (dsRNA) rescued hFUS toxicity through a double mechanism. The depleted hsrω transcript induced a decrease in hFUS mRNA levels and altered its solubility to form hFUS-LAMP1-insoluble inclusions. The combination of these events ultimately led to the titration of a hFUS molar excess and rescued toxicity. Neither proteasome, autophagy, nor lysosomes were involved in the degradation of hFUS-LAMP1 inclusions, which makes it more difficult to clarify the role of the lysosome-associated membrane protein LAMP1 and also suggests that these inclusions are specifically targeted to be a deposit as safe cytoplasmic particles (Lo Piccolo, 2017b).
These novel results revealed an evolutionarily conserved mechanism to control FUS transcripts based on lncRNA and suggest a new role for LAMP1 in the formation of cytoplasmic hFUS deposits. The present results also contribute to understanding of the pathomechanism of FUS-proteinopathies and indicate that a lncRNA-dependent mechanism may manage aggregation-prone hnRNPs such as FUS in non-toxic inclusions (Lo Piccolo, 2017b).
lncRNAs, which are a class of transcripts longer than 200 nucleotides with no or very low coding potential and similar to mRNA, but with proper specific features, are predicted to function, often preferentially, in the nervous system, in which they may play roles in mediating neuronal development, behavior, and cognitive functions. The role of lncRNAs in neurological diseases is emerging as a new and important biological aspect and the number of publications in this field has rapidly increased over the past decade. lncRNAs have been implicated in neurodegenerative diseases dependent on alterations in RNA metabolism because a large number of them are dysregulated upon the depletion of RNA-binding proteins causing FTLD and/or ALS, such as FUS and TDP43. The formation of pathological inclusions of hnRNPs, which are a hallmark of these types of diseases, may aberrantly sequester important lncRNAs into pathological RNA-containing aggregates, thereby causing a global alteration in RNA processing. From this perspective, the effects of lncRNAs in hnRNP-dependent neurodegenerative diseases are considered to be a secondary event caused by alterations in hnRNP abilities. Until now, it remained unclear whether lncRNAs induce alterations in the function of hnRNPs in order to drive toxicity (Lo Piccolo, 2017b).
Previous work has shown that the hnRNP dFUS is a new hsrω-interacting protein in Drosophila. Several other hnRNPs have the ability to bind to nuclear lncRNA hsrω to form ω-speckles, which are a specific class of nuclear bodies (NBs) involved in a wide range of cellular functions. Notably, hFUS is a component of paraspeckles (one of the major classes of NBs), which are built by the lncRNA NEAT1, and, as described above, it shows a specific ability to bind long transcripts (Lo Piccolo, 2017b).
Although global identity between hFUS and dFUS is less than 50%, the two proteins have highly similar specific domains, and the transgenic expression of hFUS may completely rescue defects caused by the loss of dFUS, thereby confirming its evolutionarily conserved function and indicating that hFUS in the fly also establishes a proper whole set of molecular interactions. However, this study found that hFUS expressed in the fly showed a cytoplasmic distribution to exclude an interaction occurring with the lncRNA hsrω limited to the nucleus. Nevertheless, it was revealed that hsrω RNAi induced a reduction in dFUS mRNA levels and negatively affected hFUS transcription. The present study drove the expression of hFUS using a Gal4 system; therefore, it will be interested to examine whether the lncRNA hsrω has the ability to bind both FUS transcripts in order to stabilize the FUS mRNAs (Lo Piccolo, 2017b).
The expression of hFUS in the fly was found to induce a depletion of endogenous dFUS, an augmentation of the lncRNA hsrω and the alteration of dFUS40:dFUS34 ratio to increase the amount of insoluble dFUS34. Moreover, as was found through fractionation studies, hFUS in the fly also showed the ability to form relative soluble aggregates. As such, when considering the FUS toxicity in fly models, all of these aspects should be taken into account. Cumulatively, the results herein reported contribute to clarify the relation of cause and effect underlying the hFUS toxicity. In fact, the current studies support the idea that a depletion of endogenous dFUS upon expression of hFUS do not lead or contribute to hFUS toxicity because flies co-expressing hFUS and hsrω dsRNA show a normal eye phenotype despite the knockdown of hsrω is able to reduce the amount of dFUS. In addition, a depletion of dFUS has been shown to cause a mild degeneration of adult eye compounds (Lo Piccolo, 2017b).
To examine whether the augmentation of hsrω observed upon hFUS expression may contribute to hFUS toxicity, the effects of hsrω overexpression was studied in a hFUS overexpression background. Interestingly, flies carrying GMR/+;hFUS/+;EP93D/+ show normal adult eye compounds with a partial loss of pigmentation. This result again strongly confirms the tight link between FUS and hsrω and supports the idea that the increased expression of hsrω upon hFUS expression in the fly may not contribute to hFUS toxicity. At the same time, these results suggest that the mechanism how hsrω knockdown can rescue the hFUS tocixity may be different from that triggered by hsrω overexpression. Further experiments are required to extend the knowledge in this regard. Notably, this study did not examine whether hsrω indirectly affects FUS through the activity of other hnRNPs, which is possible because defects in the lncRNA hsrω alter compartmentalization and the functions of diverse hnRNPs that, in turn, have roles in RNA processing (Lo Piccolo, 2017b).
Altogether the findings suggest that the hFUS amount may play a critical role in hFUS toxicity. For example, it was found that hFUS toxicity was strongest at 28 °C. Notably, the transcriptional stimulation activity of GAL4 is known to be temperature dependent to drive the highest UAS expression. Moreover, a severest eye degeneration was observed in flies carrying GMR;+ ;hFUS than in flies carrying GMR/+;hFUS/+;GFP IR/+. In light of these results, the hFUS toxicity in the fly could be a combination of events such as the formation of hFUS aggregates and the augmentation of insoluble dFUS34 which are both a result of hFUS expression. However, this study did not look at the role of insoluble dFUS34 whereby, further studies are needed in order to more fully understand the eventually impact of dFUS34 in hFUS-induced toxicity (Lo Piccolo, 2017b).
According to the current results, the first effective mechanism triggered by hsrω depletion in rescuing hFUS toxicity is considered to be a reduction in the amount of hFUS (Lo Piccolo, 2017b).
Notably, since the abundance of hFUS transcript was found high in flies co-expressing hFUS and hsrω thus showing an almost complete rescue, further studies are required to clarify the mechanism how the overexpression of hsrω can be able to induce such a similar effect. In fact, the eye phenotype observed upon co-expression of hFUS and hsrω suggest that hsrω is able to modulate a protective mechanism to control the hFUS toxicity even in the presence of an exacerbated amount of hFUS (Lo Piccolo, 2017b).
When a solubility test was performed in the hsrω RNAi background, a marked shift in hFUS from soluble to insoluble was found because hFUS only was fractionated in 8 M Urea containing-buffer. A large number of studies agree that the toxicity of hFUS does not depend on its insolubility, however in ALS and FTLD human patients, the wt FUS is immunoreactive to ubiquitin in inclusion bodies, making it difficult to firmly establish whether the solubility shift is a prerequisite for pathogenesis. Since the formation of insoluble hFUS inclusions was associated with the rescue of an aberrant eye phenotype in the present study, it is concluded that cytoplasmic hFUS inclusions are not toxic themselves, and also a question was raised regarding differences between pathological and non-pathological inclusions. Indeed, this study demonstrates that insoluble hFUS in the hsrω RNAi background was negative for ubiquitin making the hFUS inclusions of this study not corresponding to the pathological inclusions observed in human FUS-proteinopathy patients. Moreover, the hFUS inclusions detected in this study were not attacked by proteasome and even when tagged by the lysosome-associated membrane protein LAMP1, hFUS inclusions were not degraded by autophagy or lysosomes. Recent studies reported that FUS forms distinct cytoplasmic partitions similar to inclusion protein deposits (IPODs), but with specific characteristics. These new cytoplasmic compartments are referred to as Interactor Specific Compartments/Inclusions (RISCI). However, RISCI have yet to be studied in Drosophila. Nevertheless, since FUS is an aggregation-prone protein, it is speculated that a cellular mechanism to control the soluble-insoluble balance may exist in order to modulate the tendency of FUS to randomly form and sequester proteins in aberrant structures. On the other hand, in vivo studies showed that hFUS formed a specific class of non-toxic aggregates in a multi-step and RNA-dependent manner. Cytoplasmic RNA granules are generally characterized by a set of markers i.e. TIAR, G3B1, and FMRP, in stress granules (SGs), but no RISCI's marker has been characterized, so far. It was assumed that LAMP1 is one of the specific factors targeting cytoplasmic FUS to form non-toxic partitions. Since hFUS cannot directly bind to hsrω the hFUS solubility shift was considerfed to depend on a process caused by the depletion of hsrω. In addition, this study revealed that LAMP1 was up-regulated by the expression of hsrω RNAi, and, thus, speculated that altered hFUS solubility is completely or partly attributed to the interaction with LAMP1. LAMP1 is needed in order to control hFUS toxicity because its depletion in flies co-expressing hFUS mRNA and hsrω dsRNA abolishes rescue, resulting in defects with diverse severities. However, due to the augmentation of LAMP1 only in hFUS-expressing flies, it was not possible to rescue the aberrant phenotype. Two reasons have been proposed for this failure: LAMP1 may not have the ability to reduce the expression of hFUS, which supports the exacerbated amount of hFUS being the main factor inducing toxicity. Furthermore, other hsrω-dependent factors may be required for the formation of non-pathological hFUS deposits. Notably, the formation of hFUS-LAMP1 insoluble inclusions upon depletion of hsrω has been found in combination with the removal of hFUS relative soluble aggregates to support the model of hFUS aggregates play a significant role in hFUS toxicity. Therefore, the second effective mechanism triggered by hsrω depletion to rescue hFUS toxicity is considered to be the elimination of hFUS relative soluble aggregates trough the formation of LAMP1-targeted hFUS insoluble inclusions (Lo Piccolo, 2017b).
The novel results of the present study suggest a new scenario; a lncRNA-dependent mechanism may control the formation of non-toxic FUS inclusions. Under these conditions, the mis-regulation of some lncRNAs may not only be a consequence of FUS aggregate formation, as previously proposed, it may also be an instigating event of FUS-proteinopathies such as alterations in FUS expression and specific targeting/packaging into harmless deposits (Lo Piccolo, 2017b).
Defective RNA metabolism is common pathogenic mechanisms involved in neurological disorders. Indeed, a conspicuous feature of some neurodegenerative diseases is the loss of nuclear activities of RNA-binding proteins (RBPs) like Fused in sarcoma (FUS) and eventually, their accumulation in cytoplasmic proteinaceous inclusions. Long non-coding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes, including neurological disorders. A subset of these lncRNAs is the core of nuclear bodies (NBs), which are the sites of RNA processing and sequestration of specific ribonucleoproteins (RNPs) complexes. In Drosophila melanogaster the lncRNA hsromega is the architectural RNA (arcRNA) of the NB omega speckles (omega-speckles). This study shows that the neuron-specific and motor neuron-specific knockdown of hsromega impairs locomotion in larval and adult flies and induces anatomical defects in presynaptic terminals of motor neurons, suggesting a novel role of arcRNA hsromega in development of neuromuscular junctions. Since RBPs are recognized as important regulators of neuronal activities, to examine the molecular mechanism of such neurodegeneration, interaction between hsromega and Drosophila orthologue of human FUS (dFUS: Cabeza). Strictly, it was found that dFUS genetically and physically interacts with the arcRNA hsromega was examined. Moreover, it was revealed that a fine regulation of gene expression occurs between hsromega and dFUS and surprisingly, it was uncovered that depletion of hsromega affects the sub-cellular compartmentalization of dFUS thus, enhancing its cytoplasmic localization and inducing its loss of nuclear function. A proposed model shows the role of arcRNA in diseases affecting the nervous system and in particular it elucidates the molecular mechanism underlying the loss of dFUS nuclear function in the absence of its mutations. These new findings could provide new insights into the pathogenesis of neurodegenerative disease dependent on mis-function or mis-localization of aggregation prone RNA binding proteins like FUS in Amyotrophic Lateral Sclerosis (Lo Piccolo, 2017a).
Neuron-specific knockdown of the dFIG4 gene, a Drosophila homologue of human FIG4 and one of the causative genes for Charcot-Marie-Tooth disease (CMT), reduces the locomotive abilities of adult flies, as well as causing defects at neuromuscular junctions, such as reduced synaptic branch length in presynaptic terminals of the motor neurons in third instar larvae. Eye imaginal disc-specific knockdown of dFIG4 induces abnormal morphology of the adult compound eye, the rough eye phenotype. A modifier screening of the dFIG4 knockdown-induced rough eye phenotype was carried out using a set of chromosomal deficiency lines on the second chromosome. By genetic screening, 9 and 15 chromosomal regions were detected whose deletions either suppressed or enhanced the rough eye phenotype induced by the dFIG4 knockdown. By further genetic screening with mutants of individual genes in one of these chromosomal regions, the gene CR18854 was identified that suppressed the rough eye phenotype and the loss-of-cone cell phenotype. The CR18854 gene encodes a long non-coding RNA (lncRNA) consisting of 2566 bases. Mutation and knockdown of CR18854 patially suppressed the enlarged lysosome phenotype induced by Fat body-specific knockdown of dFIG4. Further characterization of CR18854, and a few other lncRNAs in relation to dFIG4 in neuron, using neuron-specific dFIG4 knockdown flies indicated a genetic link between the dFIG4 gene and lncRNAs including CR18854 and hsromega. Data was obtained indicating genetic interaction between CR18854 and Cabeza, a Drosophila homologue of human FUS, which is one of the causing genes for amyotrophic lateral sclerosis (ALS). These results suggest that lncRNAs such as CR18854 and hsromega are involved in a common pathway in CMT and ALS pathogenesis (Muraoka, 2018).
Hrp36/Hrb87F is one of the most abundant and well-characterized hnRNP A homolog in Drosophila and is shown to have roles in regulation of alternative splicing, heterochromatin formation, neurodegeneration, etc. Yet, hrp36 null individuals were reported to be viable and without any apparent phenotype, presumably because of overlapping functions provided by Hrp38 and related proteins. This study shows that loss of both copies of hrp36 gene slows down development with significant reduction in adult life span, decreased female fecundity and high sensitivity to starvation and thermal stresses. In the absence of Hrp36, the nucleoplasmic omega speckles are nearly completely disrupted. The levels of nuclear matrix protein Megator and the chromatin remodeller ISWI are significantly elevated in principal cells of larval Malpighian tubules, which also display additional endoreplication cycles and good polytene chromosomes. It is suggested that besides the non-coding hsr omega-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsr omega locus is affected in hrp36 null cells, which may be one of the reasons for their high sensitivity to cell stress. Therefore, in spite of the functional redundancy provided by Hrp38, Hrp36 is essential for normal development and for survival under conditions of stress (Singh, 2012).
The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. This study provides multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, it was found that the organization of nucleoplasmic omega speckles depends on ISWI function. These findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA (Onorati, 2011).
Factors that coordinate nuclear activities occurring on chromatin and the nucleoplasmic compartments remain unidentified and uncharacterized. Therefore, an important open question in nuclear organization field is how nuclear speckles localize and organize themselves near transcriptionally active genes to cross talk with chromatin factors for processing of the nascent RNAs. These data indicate that ISWI may provide a functional 'bridge' between chromatin and nuclear speckle compartments. Indeed, ISWI can directly or indirectly contact the omega speckles in intact nuclei, through hsrω-n ncRNA or some of the associated hnRNPs. Confocal analysis suggested a functional 'bridge' between a chromatin factor (ISWI) and nucleoplasmic omega speckle components (hsrω ncRNA and hnRNPs). However, not all omega speckles show partial overlap with ISWI. Indeed, these molecular 'bridges' between chromatin and nucleoplasm are probably transient, since time-lapse movies on live cells with fluorescently tagged chromatin and omega-speckle components clearly show very high mobility of these speckles, which probably may explain the absence of classic co-localization between ISWI and omega speckle components. (Onorati, 2011).
The observed direct physical interaction between ISWI and hsrω-n ncRNA together with the stimulation of ISWI-ATPase activity in light of the partial overlap revealed by confocal microscopy suggests that ISWI may interact with hsrω-forming speckles only transiently, probably to help the hsrω ncRNA to properly associate with or release the various omega speckle-associated hnRNPs. Loss of ISWI may impair the correct maturation, organization or localization of omega speckles resulting in an observed omega 'trail' phenotype (Onorati, 2011).
The data also provide a possible explanation for the suppression of ISWI defects by hsrω-RNAi. In ISWI mutants carrying normal levels of hsrω transcripts, the limited maternally derived ISWI is shared between chromatin remodelling and omega speckle organization reactions so that its sub-threshold levels in either compartments severely compromises both functions. However, when hsrω transcript levels are reduced by RNAi in ISWI null background, most of the maternal ISWI may become available for chromatin remodelling reactions, so that a minimal threshold level of chromosome organization can be achieved. This would permit initiation of close to normal developmental gene activity programs resulting in suppression of the ISWI eye and chromosome defects or in the postponement of the larval lethality to pupal stage. Additionally, it is known that when hsrω ncRNA is down-regulated through RNAi, levels of free hnRNPs and other chromatin factors (i.e., CBP) are also elevated. Therefore, the possibility that these changes may also counteract ISWI defects by as yet unknown mechanisms cannot be excluded (Onorati, 2011).
This work provides the first example of modulation of an ATP-dependent chromatin remodeler by a ncRNA, and is the first in vivo and in vitro demonstration of a role of chromatin remodeler in organization of a nuclear compartment. However, the mechanism underlying stimulation of the ATPase activity of ISWI by the hsrω-n ncRNA, which may facilitate the organization of omega speckles, remains to be understood. Given the evolutionary derivation of the ISWI ATPase-domain from RNA-helicase-domains, a provocative hypothesis is that ISWI could 'remodel' speckles by structurally helping the assembly or release of specific hnRNPs with the hsrω-n ncRNA to generate mature omega speckles. Chromatin remodelers, nuclear speckles and their associated long ncRNAs are emerging as essential components of gene regulatory networks, and their deregulation may underlie complex diseases. The functional homology of the human noncoding sat III transcripts with the Drosophila hsrω ncRNA (Jolly, 2006), highlights the relevance and translational significance of studies unraveling the functional connections between ncRNA-containing nuclear compartments and chromatin remodelers. (Onorati, 2011).
Following earlier reports on modulation of poly(Q) toxicity in Drosophila by the developmentally active and stress-inducible noncoding Hsromega gene (Heat shock RNA ω), possible mediators of this modulation were investigated. RNAi-mediated downregulation of the large nuclear hsromega-n transcript, which organizes the nucleoplasmic omega speckles, suppresses the enhancement of poly(Q) toxicity brought about by reduced availability of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb87F and of the transcriptional regulator, cAMP response element binding (CREB) binding protein (CBP). Levels of CBP RNA and protein are reciprocally affected by hsromega transcript levels in eye disc cells. The data suggest that CBP and hnRNPs like Hrb57A and Hrb87F physically interact with each other. In addition, downregulation of hsromega transcripts partially rescues eye damage following compromised proteasome activity, while overexpression of hsromega and/or poly(Q) proteins disrupts the proteasomal activity. Rescue of poly(Q) toxicity by hsromega-RNAi requires normal proteasomal function. It is suggested that hsromega-RNAi suppresses poly(Q) toxicity by elevating cellular levels of CBP, by enhancing proteasome-mediated clearance of the pathogenic poly(Q) aggregates, and by inhibiting induced apoptosis. The direct and indirect interactions of the hsromega transcripts with a variety of regulatory proteins like hnRNPs, CBP, proteasome, Drosophila inhibitor of apoptosis protein 1 (DIAP1), etc., reinforce the view that the noncoding hsromega RNA functions as a 'hub' in cellular networks to maintain homeostasis by coordinating the functional availability of crucial cellular regulatory proteins (Mallik, 2010).
Earlier studies have shown that while overexpression of the noncoding hsrω transcripts enhanced (Sengupta, 2006), reducing the cellular levels of these transcripts through RNAi suppressed the neurodegeneration caused by mutant proteins with expanded poly(Q) stretches (Mallik, 2009a). As shown earlier (Mallik, 2009a), expression of the hsrω-RNAi transgene had no effect on poly(Q) transcription but it diminished/eliminated the source of toxicity by inhibiting formation of the inclusion bodies (IBs) and by enhancing clearance of the mutant poly(Q) proteins. The present study provides useful insights into the possible mechanisms through which these noncoding transcripts modulate cellular toxicity of the mutant poly(Q) proteins (Mallik, 2010).
Studies in a variety of poly(Q) model systems have reported that many essential cellular proteins, e.g., transcription factors like CBP, TBP; chaperone proteins, etc., are sequestered by the expanded poly(Q) proteins. In agreement with earlier reports, the present study shows that the poly(Q) damage is enhanced by functional depletion of hnRNPs, CBP, or proteasome components because of expression of dominant-negative mutants or RNAi or null mutations. hsrω-RNAi substantially rescued the poly(Q) toxicity even when additional damage was caused by the presence of mutant alleles of Hrb87F or CBP. In contrast, compromised proteasome activity affected the rescue of poly(Q) damage by hsrω-RNAi (Mallik, 2010).
It is significant that while complete absence of Hrb87F does not affect normal development of Drosophila melanogaster, ~20% reduction in cellular levels of Hrb87F, as seen in the Df(3R)Hrb87F/+ eye discs, resulted in a significant enhancement in the poly(Q) eye phenotype. As noted earlier, the mutant poly(Q) proteins deplete the functional availability of many essential proteins and thus disrupt cellular homeostasis. Therefore, even a 20% depletion of the otherwise dispensable Hrb87F exaggerates the poly(Q) toxicity. Mallik (2009a) has shown that hsrω-RNAi results in disappearance of the omega speckles so that the various proteins, including Hrb87F, sequestered in them become available in the soluble cellular pool. The increased availability of such essential proteins in the functional pool following hsrω-RNAi compensates not only for the genetic deficiency of Hrb87F but also for the functional depletion of this and other proteins by the poly(Q) IBs. This finds support in the fact that targeted overexpression of hnRNP A2/B1 and its Drosophila homologs, Hrb87F and Hrb98DE, suppresses CGG repeat-induced neurodegeneration in the FXTAS fly model (Sofola, 2007). It has recently been shown (Ji, 2009) that poly(ADP) ribosylation and deglycosylation of hnRNPs modulate their activity and their binding with the hsrω transcripts; it was suggested that only nonribosylated hnRNPs can be sequestered by these transcripts. It is, therefore, likely that the release of hnRNPs from the omega speckles following hsrω-RNAi provides for a greater pool of the hnRNPs being available for ribosylation and thus activity (Mallik, 2010).
CBP is one of the important regulators of chromatin structure and transcription and its sequestration by the mutant poly(Q) proteins is believed to be a major cause for neurodegeneration (Li, 2004; Bae, 2005). It is also reported that overexpressing CBP or enhancing its activity suppresses poly(Q) IB formation and neurodegeneration (Taylor, 2003). The findings that developmental defects in eyes caused by expression of dominant-negative forms of CBP or by its depletion through RNAi are rescued by hsrω-RNAi clearly show that the hsrω transcripts can modulate CBP metabolism in eye disc cells. This possibility is confirmed by the finding that levels of CBP transcripts and that of the CBP protein are elevated following hsrω-RNAi and are lowered by hsrω overexpression (Mallik, 2010).
The observations that cellular distributions of hnRNPs, like Hrb87F and Hrb57A, partially overlap with that of CBP and that these proteins are co-immunoprecipitated suggest that CBP interacts with Hrb57A and Hrb87F. Downregulation of hsrω-n RNA results in disappearance of the omega speckles and redistribution of the hnRNPs (Mallik, 2009a). In view of the physical association of these proteins, it is speculated that the enhanced availability of hnRNPs in the diffuse cellular pool may pull more CBP into the diffuse fraction so that a greater amount of CBP becomes available for activity rather than remaining stored/sequestered. Additionally, caspase-6-mediated cleavage and degradation of CBP followed by a subsequent decrease in histone acetylation, another critical step common to several neuropathologies, may also be inhibited by hsrω-RNAi since other studies showed that hsrω-RNAi inhibits caspase activity through stabilization of DIAP1 via its interaction with Hrb57A (Mallik, 2009b). The levels of hsrω-n transcripts may also affect CBP mRNA levels through the variety of RNA-processing and transcription factors that directly or indirectly associate with the hsrω transcripts. As reported earlier, the net increase in CBP levels, following hsrω-RNAi, would inhibit formation of poly(Q) IBs and restore the histone acetylation homeostasis (Mallik, 2010).
It is remarkable that while hsrω-RNAi suppressed the eye phenotypes resulting from expression of CBP-FL AD or CBP RNAi or CBP DeltaNZK, it failed to rescue the lethality or the eye damage following expression of CBP DeltaQ or CBP DeltaBHQ, respectively. This indicates that the transactivation domain of CBP is required for the suppressive action of hsrω-n RNAi. It is likely that the hnRNPs like Hrb87F, Hrb57A, etc., interact with CBP through its Q domain so that the hnRNPs released by disappearance of the omega speckles following hsrω-RNAi fail to compensate the damage caused by expression of dominant-negative CBP DeltaQ or CBP DeltaBHQ. Further studies are required to understand the mechanism(s) of these interactions (Mallik, 2010).
These studies also reveal interaction of ubiquitin proteasome pathway (UPP) with hsrω transcripts and an important role of this interaction in the modulation of poly(Q) toxicity. Restoration of the eye phenotype following targeted disruption of the normal proteasomal activity by hsrω-RNAi indicates that the proteasome activity improves when levels of these noncoding transcripts are reduced. This is also confirmed by the direct demonstration, through the GFP-reporter expression, that the intrinsic UPP is compromised in cells overexpressing hsrω. The finding that proteasome activity is impaired in 127Q-expressing flies is consistent with earlier reports that poly(Q) toxicity in vivo is enhanced by proteasome mutations or by inhibitors of proteasome activity. It is significant that hsrω-RNAi ameliorated the proteasomal dysfunction due to poly(Q) expression, since the proteasome-GFP reporter expression was very low in cells coexpressing poly(Q) and hsrω-RNAi. Restoration of proteasome function in mutant poly(Q)-expressing cells is thus an additional pathway through which hsrω-RNAi suppresses the neurodegeneration. This finds further support in the observation that when the endogenous UPP function is intrinsically compromised by expression of dominant-negative mutants, hsrω-RNAi is no longer as effective in suppressing the poly(Q) damage as in cells with normal proteasome function. The mechanism(s) through which the hsrω transcripts regulate UPP pathways remain to be understood (Mallik, 2010).
Many of the poly(Q) proteins involved in CAG repeat expansion disorders contain caspase consensus cleavage sites and caspase-mediated cleavage of the mutant protein appears necessary for pathogenesis (Evert, 2000). Inhibition of activity of caspases like caspase-1, caspase-3, or caspase-8 or alteration of the caspase cleavage sites in the mutated protein delays and reduces the expanded poly(Q) protein pathogenicity. Other studies show that in cells in which apoptosis is ectopically induced, hsrω-RNAi stabilizes DIAP1 through enhanced association with Hrb57A (Mallik, 2009b). Elevated levels of DIAP1 inhibit caspase activity and thus apoptosis. Further, expression of expanded poly(Q) proteins brings about hyperactivation of JNK, which contributes to neuronal dysfunction and cell death in neurodegenerative disorders. Significantly, hsrω-RNAi suppresses activation of the JNK pathway also (Mallik, 2009b). Inhibition of caspase and JNK activities thus appear to be other paths through which hsrω-RNAi suppresses the poly(Q) toxicity in the fly models (Mallik, 2010).
In summary, it is suggested that hsrω-RNAi suppresses poly(Q) toxicity by modulating several components involved in the pathogenesis of these debilitating diseases. First, hsrω-RNAi enhances the availability of hnRNPs and CBP in functional pools. This in turn would suppress IB formation and restore histone acetylation and transcriptional regulation in cells expressing the mutant poly(Q) proteins. Second, the proteasomal activity is improved when hsrω RNA levels are reduced and this helps the cells to get rid of toxic proteins. Third, the release of hnRNPs from omega speckles following hsrω-RNAi stabilizes DIAP1 (Mallik, 2009b), resulting in inhibition of apoptosis so that neuronal cells, that otherwise would have died, survive. Additionally, in view of the above noted role of JNK in poly(Q) damage, the suppression of JNK activation in eye disc cells following hsrω-RNAi (Mallik, 2009b) may also contribute to amelioration of the poly(Q) damage. Further, the hsrω transcripts are known to interact with several other proteins, including Hsp90, and therefore, it remains possible that other network effects also contribute to the observed suppression of the poly(Q) damage. The observed pleiotropic effects reflect involvement and, therefore, critical importance of the hsrω noncoding transcripts in cellular homeostasis. Since most of the wild-type poly(Q) proteins, whose mutations result in neurodegeneration, are themselves involved in diverse regulatory processes, alterations in the noncoding hsrω transcript pool can be expected to bring about unpredictable and divergent consequences in cells with genetically compromised regulation. These transcripts apparently function as hubs for coordination of several cellular networks 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 the same cell under different conditions. The multipronged action of these noncoding transcripts also provides a new paradigm for a therapeutic target for the human poly(Q) disorders (Mallik, 2010).
While hundreds of genes have recently been implicated in an organism's response to thermal stress, insight into the cellular and physiological mechanisms affected by these genes has advanced to a lesser extent. This study focused on an enigmatic Drosophila heat stress RNA gene, hsr-omega, which encodes two RNA transcripts that are constitutively expressed in almost all developing and adult tissues, omega-n in the nucleus and omega-c in the cytoplasm; both being readily induced to high levels by mild heat stress. Three hsr-omega mutant lines were derived via imprecise P-element excision, and they were characterised for changes in expression, in both the presence and absence of heat stress. Viability estimates indicate that a low level of omega-n is required for normal development. Consistent with the model of omega-n as a negative regulator of intron-processed mRNA levels the mutants displayed a 1.5-fold increase in rates of protein synthesis measured in ovarian tissue in the absence of heat stress, a result suggesting that an important function of hsr-omega is the modulation of general protein synthesis. The mutants had little effect on two measures commonly used to assess heat tolerance, heat-knockdown time and heat hardening ability, suggesting that more subtle heat-related fitness components need to be examined for effects of these mutations (Johnson, 2011).
Drosophila melanogaster occurs in diverse climatic regions and shows opposing clinal changes in resistance to heat and resistance to cold along a 3000 km latitudinal transect on the eastern coast of Australia. This study reports on variation at a polymorphic 8 bp-indel site in the heat shock hsr-omega gene that maps to the right arm of chromosome 3. The frequency of the genetic element marked by the L form of the gene was strongly and positively associated with latitude along this transect, and latitudinal differences in L frequency were robustly associated with latitudinal differences in maximum temperature for the hottest month. On a genetic background mixed for genes from each end of the cline a set of 10 lines was derived, five of which were fixed for the L marker, the absence of In(3R)P and 12 kb of repeats at a second polymorphic site at the 3' end of hsr-omega, and five that were fixed for the S marker, In(3R)P and 15 kb of hsr-omega repeats. For two different measures of heat tolerance S lines outperformed L lines, and for two different measures of cold tolerance L lines outperformed S lines. These data suggest that an element on the right arm of chromosome 3, possibly In(3R)P, confers heat resistance but carries the trade-off of also conferring susceptibility to cold. This element occurs at high frequency near the equator. The alternate element on the other hand, at high frequency at temperate latitudes, confers cold resistance at the cost of heat susceptibility (Anderson, 2003).
Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsromega-n) transcripts were present in the form of many small speckles. These speckles, which were named 'omega speckles', were distributed in the interchromatin space in close proximity to the chromatin. The only chromosomal site where hsromega-n transcripts localized was the 93D locus or the hsromega gene itself. The number of nucleoplasmic speckles varied in different cell types. Heat shock, which inhibits general chromosomal transcription, caused the individual speckles to coalesce into larger but fewer clusters. In extreme cases, only a single large cluster of hsromega-n transcripts localizing to the hsromega locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuclear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and S5 revealed that, in all cell types, all the hnRNPs gave a diffuse staining of chromatin areas and in addition, were present as large numbers of speckles. Colocalization studies revealed an absolute colocalization of the hnRNPs and the omegaspeckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsromega-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsromega transcripts. The omegaspeckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the ω speckles. The speckled distribution of hnRNPs was completely disrupted in hsromega nullosomics. It is concluded that the hsromega-n transcripts play essential structural and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus (Prasanth, 2000).
Inducible heat shock genes are considered a major component of the molecular mechanisms that confer cellular protection against a variety of environmental stresses, in particular high temperature extremes. This study has tested the association between expression of the heat shock RNA gene hsr-omega and thermoresistance by generating thermoresistant lines of Drosophila melanogaster after application of two distinct regimes of laboratory selection. One set of lines was selected for resistance to knockdown by heat stress and the other was similarly selected but before selection a mild heat exposure known to increase resistance (heat hardening) was applied. A cross between resistant and susceptible lines confirmed earlier observation that increased thermal tolerance cosegregates with allelic variation in the hsr-omega gene. This cosegregating variation is attributed largely to two haplotype groups. Using quantitative reverse transcription-PCR, evidence was found for divergent phenotypic responses in the two selection regimes, involving both structural and regulatory changes in hsr-omega. Lines selected after hardening showed increased levels of the cytoplasmic transcript but decreased levels of the nuclear transcript. Lines selected without hardening showed decreased levels of the cytoplasmic transcript. The allelic frequency changes at hsr-omega could not by themselves account for the altered transcription patterns. These results support the idea that the functional RNA molecules transcribed from hsr-omega are an important and polymorphic regulatory component of an insect thermoresistance phenotype (McKechnie, 1998).
Salivary glands of Drosophila larvae were treated in vitro with benzamide or with a homogenate of heat shocked glands to specifically induce high transcriptional activity of the 93D puff. The newly synthesized 14C-amino acids labelled polypeptides in the treated and sister control glands were analysed by polyacrylamide gel electrophoresis, followed by gel autoradiography. The protein synthesis patterns in the treated glands in either case remain the same as in control glands. No novel polypeptide was seen which could be correlated with the high induced transcriptional activity of the 93D puff. This suggests that the 93D transcript/s is/are probably not translated (Lakhotia, 1982).
Search PubMed for articles about Drosophila hsromega
Anderson, A. R., Collinge, J. E., Hoffmann, A. A., Kellett, M. and McKechnie, S. W. (2003). Thermal tolerance trade-offs associated with the right arm of chromosome 3 and marked by the hsr-omega gene in Drosophila melanogaster. Heredity (Edinb) 90(2): 195-202. PubMed ID: 12634827
Johnson, T. K., Cockerell, F. E. and McKechnie, S. W. (2011). Transcripts from the Drosophila heat-shock gene hsr-omega influence rates of protein synthesis but hardly affect resistance to heat knockdown. Mol Genet Genomics 285(4): 313-323. PubMed ID: 21399957
Jolly, C. and Lakhotia, S. C. (2006). Human sat III and Drosophila hsr omega transcripts: a common paradigm for regulation of nuclear RNA processing in stressed cells. Nucleic Acids Res 34(19): 5508-5514. PubMed ID: 17020918
Lakhotia, S. C. and Mukherjee, T. (1982). Absence of novel translation products in relation to induced activity of the 93D puff in Drosophila melanogaster. Chromosoma 85(3): 369-374. PubMed ID: 6811224
Lakhotia, S. C. (2012a). Long non-coding RNAs coordinate cellular responses to stress. Wiley Interdiscip Rev RNA 3(6): 779-796. PubMed ID: 22976942
Lakhotia, S. C., Mallik, M., Singh, A. K. and Ray, M. (2012a). The large noncoding hsromega-n transcripts are essential for thermotolerance and remobilization of hnRNPs, HP1 and RNA polymerase II during recovery from heat shock in Drosophila. Chromosoma 121(1): 49-70. PubMed ID: 21913129
Lo Piccolo, L. and Yamaguchi, M. (2017a). RNAi of arcRNA hsromega affects sub-cellular localization of Drosophila FUS to drive neurodiseases. Exp Neurol 292: 125-134. PubMed ID: 28342748
Lo Piccolo, L., Jantrapirom, S., Nagai, Y. and Yamaguchi, M. (2017b). FUS toxicity is rescued by the modulation of lncRNA hsromega expression in Drosophila melanogaster. Sci Rep 7(1): 15660. PubMed ID: 29142303
Lo Piccolo, L., Bonaccorso, R., Attardi, A., Li Greci, L., Romano, G., Sollazzo, M., Giurato, G., Ingrassia, A. M. R., Feiguin, F., Corona, D. F. V. and Onorati, M. C. (2018). Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43. Int J Mol Sci 19(4). PubMed ID: 29617352
Mallik, M. and Lakhotia, S. C. (2009a). RNAi for the large non-coding hsromega transcripts suppresses polyglutamine pathogenesis in Drosophila models. RNA Biol 6(4): 464-478. PubMed ID: 19667761
Mallik, M. and Lakhotia, S. C. (2009b). The developmentally active and stress-inducible noncoding hsromega gene is a novel regulator of apoptosis in Drosophila. Genetics 183(3): 831-852. PubMed ID: 19737742
Mallik, M. and Lakhotia, S. C. (2010). Improved activities of CREB binding protein, heterogeneous nuclear ribonucleoproteins and proteasome following downregulation of noncoding hsromega transcripts help suppress poly(Q) pathogenesis in fly models. Genetics 184(4): 927-45. PubMed Citation: 20065067
McKechnie, S. W., Halford, M. M., McColl, G. and Hoffmann, A. A. (1998). Both allelic variation and expression of nuclear and cytoplasmic transcripts of Hsr-omega are closely associated with thermal phenotype in Drosophila. Proc Natl Acad Sci U S A 95(5): 2423-2428. PubMed ID: 9482901
Muraoka, Y., Nakamura, A., Tanaka, R., Suda, K., Azuma, Y., Kushimura, Y., Lo Piccolo, L., Yoshida, H., Mizuta, I., Tokuda, T., Mizuno, T., Nakagawa, M. and Yamaguchi, M. (2018). Genetic screening of the genes interacting with Drosophila FIG4 identified a novel link between CMT-causing gene and long noncoding RNAs. Exp Neurol 310: 1-13. PubMed ID: 30165075
Onorati, M. C., Lazzaro, S., Mallik, M., Ingrassia, A. M., Carreca, A. P., Singh, A. K., Chaturvedi, D. P., Lakhotia, S. C. and Corona, D. F. (2011). The ISWI chromatin remodeler organizes the hsromega ncRNA-containing omega speckle nuclear compartments. PLoS Genet 7(5): e1002096. PubMed ID: 21637796
Place, R. F. and Noonan, E. J. (2014). Non-coding RNAs turn up the heat: an emerging layer of novel regulators in the mammalian heat shock response. Cell Stress Chaperones 19(2): 159-172. PubMed ID: 24002685
Prasanth, K. V., Rajendra, T. K., Lal, A. K. and Lakhotia, S. C. (2000). Omega speckles - a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila. J Cell Sci 113 Pt 19: 3485-3497. PubMed ID: 10984439
Roberts, S. P. and Feder, M. E. (1999). Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster. Oecologia 121(3): 323-329. PubMed ID: 28308320
Singh, A. K. and Lakhotia, S. C. (2012). The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster. J Biosci 37(4): 659-678. PubMed ID: 22922191
Singh, A. K. and Lakhotia, S. C. (2015). Dynamics of hnRNPs and omega speckles in normal and heat shocked live cell nuclei of Drosophila melanogaster. Chromosoma 124(3): 367-383. PubMed ID: 25663367
Singh, A. K. and Lakhotia, S. C. (2016). Expression of hsromega-RNAi transgene prior to heat shock specifically compromises accumulation of heat shock-induced Hsp70 in Drosophila melanogaster. Cell Stress Chaperones 21(1): 105-120. PubMed ID: 26386576
date revised: 20 November 2019
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