Dysregulation of core components of SCF complex in poly-glutamine disorders.

Poly-glutamine (polyQ) diseases are neurodegenerative disorders characterised by expanded CAG repeats in the causative genes whose proteins form inclusion bodies. Various E3 ubiquitin ligases are implicated in neurodegenerative disorders. We report that dysfunction of the SCF (Skp1-Cul1-F-box protein) complex, one of the most well-characterised ubiquitin ligases, is associated with pathology in polyQ diseases like Huntington's disease (HD) and Machado-Joseph disease (MJD). We found that Cullin1 (Cul1) and Skp1, core components of the SCF complex, are reduced in HD mice brain. A reduction in Cul1 levels was also observed in cellular HD model and fly models of both HD and MJD. We show that Cul1 is able to genetically modify mutant huntingtin aggregates because its silencing results in increased aggregate load in cultured cells. Moreover, we demonstrate that silencing dCul1 and dSkp1 in Drosophila results in increased aggregate load and enhanced polyQ-induced toxicity. Our results imply that reduced levels of SCF complex might contribute to polyQ disease pathology.

Hsp60D is essential for caspase-mediated induced apoptosis in Drosophila melanogaster.

Apart from their roles as chaperones, heat shock proteins are involved in other vital activities including apoptosis with mammalian Hsp60 being ascribed proapoptotic as well as antiapoptotic roles. Using conditional RNAi or overexpression of Hsp60D, a member of the Hsp60 family in Drosophila melanogaster, we show that the downregulation of this protein blocks caspase-dependent induced apoptosis. GMR-Gal4-driven RNAi for Hsp60D in developing eyes dominantly suppressed cell death caused by expression of Reaper, Hid, or Grim (RHG), the key activators of canonical cell death pathway. Likewise, Hsp60D-RNAi rescued cell death induced by GMR-Gal4-directed expression of full-length and activated DRONC. Overexpression of Hsp60D enhanced cell death induced either by directed expression of RHG or DRONC. However, the downregulation of Hsp60D failed to suppress apoptosis caused by unguarded caspases in DIAP1-RNAi flies. Furthermore, in DIAP1-RNAi background, Hsp60D-RNAi also failed to inhibit apoptosis induced by RHG expression. The Hsp60 and DIAP1 show diffuse and distinct granular overlapping distributions in the photoreceptor cells with the bulk of both proteins being outside the mitochondria. Depletion of either of these proteins disrupts the granular distribution of the other. We suggest that in the absence of Hsp60D, DIAP1 is unable to dissociate from effecter and executioner caspases, which thus remain inactive.

Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster.

Hsp60C gene of Drosophila melanogaster shows a dynamic spatiotemporal expression during oogenesis and seems to contribute bulk of the Hsp60 family proteins in ovarioles. Hsp60 distribution overlaps with that of F-actin-rich membranes/structures in follicle, nurse, and egg cells throughout oogenesis. Skeletal muscle fibers associated with ovarioles and in other parts of the body show patterned location of Hsp60 in A-bands. During stages 11-12, Hsp60 accumulates at junctions of nurse cells and oocyte, where a new microtubule organizing center is known to develop. A recessive hypomorph allele, Hsp60C1 causes complete sterility of the rare surviving homozygous adults. Their egg chambers show very little Hsp60C transcripts or Hsp60 protein. Beginning at stages 6-7, Hsp60C1 chambers show a disorganized follicle cell layer with poor cell adhesion in addition to abnormal organization of F-actin and other cytoskeletal structures in follicle, nurse, and egg cells. Additionally, expression and localizations of Hrb98DE, Squid, and Gurken proteins in nurse cells and oocyte are also severely affected. Hsp60C1 homozygous follicle cell clones in Hsp60C1/+ ovarioles show disruptions in follicle epithelial and cytoskeleton arrangements. Likewise, egg chambers with Hsp60C1 homozygous germline clones in Hsp60C1/+ flies show abnormal oogenesis. Our results provide the first evidence for an essential role of Hsp60C in Drosophila oogenesis, especially in organization and maintenance of cytoskeletal and cell adhesion components.

Altered expressions of the noncoding hsromega gene enhances poly-Q-induced neurotoxicity in Drosophila.

In an earlier report two P-transposon insertion alleles of the noncoding hsromega gene, hsromega(05241) and P292 were shown to enhance neurodegeneration caused by expression of ataxin-1 protein with expanded poly-Q in a Drosophila model. In present study, we examined the possible relation between hsromega gene expression and toxicity due to poly-Q pathogenesis. The Drosophila hsromega gene produces several noncoding transcripts in almost all cell types, of which the >10 kb long hsromega-n transcript organizes heterogeneous RNA binding (hnRNPs) and related proteins as nucleoplasmic omega speckles. We show that P insertion alleles of the hsromega gene, which cause its overexpression, dominantly enhance neurodegeneration in fly eyes expressing either expanded poly-Q (127Q) or mutant huntingtin protein. Null allele of Hrb87F gene, encoding hnRNPA1, and a novel gene's mutant allele (l(3)pl10(R)), which affects the omega speckles, also dominantly enhance 127Q-induced neurodegeneration. The hsromega-n transcripts or the hnRNPs do not colocalize with the poly-Q nuclear inclusion bodies, neither in hsromega wild type, nor in hsromega mutant background. However, the levels of poly-Q and Hsp70 were significantly higher in hsromega mutant eye discs. Sequestration of hnRNPs and other related RNA-binding proteins by overexpression of hsromega transcripts in hsromega(05241) or in l(3)pl10(R) background or the reduced levels of Hrb87F protein seem to affect nuclear RNA metabolism, thus enhancing the toxicity due to poly-Q expansion.

Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions.

In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae I(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.

Tissue- and development-specific induction and turnover of hsp70 transcripts from loci 87A and 87C after heat shock and during recovery in Drosophila melanogaster.

The haploid genome of Drosophila melanogaster normally carries at least five nearly identical copies of heat-shock-inducible hsp70 genes, two copies at the 87A7 and three copies at the 87C1 chromosome sites. We used in situ hybridization of the cDNA, which hybridizes with transcripts of all five hsp70 genes, and of two 3' untranslated region (3'UTR; specific for the 87A7- and 87C1-type hsp70 transcripts) riboprobes to cellular RNA to examine whether all these copies were similarly induced by heat shock in different cell types of D. melanogaster. Our results revealed remarkable differences not only in the heat-shock-inducibility of the hsp70 genes at the 87A7 and 87C1 loci, but also in their post-transcriptional metabolism, such as the stability of the transcripts and of their 3'UTRs in different cell types in developing embryos and in larval and adult tissues. Our results also revealed the constitutive presence of the heat-shock-inducible form of Hsp70 in a subset of late spermatogonial cells from the second-instar larval stage onwards. We suggest that the multiple copies of the stress-inducible hsp70 genes do not exist in the genome of D. melanogaster only to produce large amounts of the Hsp70 rapidly and at short notice, but that they are specifically regulated in a developmental-stage-specific manner. It is likely that the cost/benefit ratio of not producing or of producing a defined amount of Hsp70 under stress conditions varies for different cell types and under different physiological conditions and, accordingly, specific regulatory mechanisms operating at the transcriptional and post-transcriptional levels have evolved.

Developmental regulation and complex organization of the promoter of the non-coding hsr(omega) gene of Drosophila melanogaster.

The nucleus-limited large non-coding hsr(omega)-n RNA product of the 93D or the hsr(omega) gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsr(omega) 05241 allele due to insertion of a P-LacZ-rosy+ transposon at -130 bp position of the hsr(omega) promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsr(omega) promoter upstream of the LacZ reporter. The hsr(omega) gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsr(omega) 05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsr(omega) transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsr(omega) RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsr(omega) gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.

Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

Omega speckles - a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila.

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 we name '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 &ohgr; speckles. The speckled distribution of hnRNPs was completely disrupted in hsromega nullosomics. We conclude 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.

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects.

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.

Genetic mapping of the amide response element(s) of the hsr omega locus of Drosophila melanogaster.

Small chromosomal deletions [Df(3R)eR-1 and Df(3R)eP] with intact hsromega transcription units but with variable deletions of the upstream region were used to map the upstream regions that regulate heat shock and amide responsivity of the 93D puff (hsromega locus) in salivary glands of late third instar larvae of Drosophila melanogaster. The Df(3R)eP deletion, generated by a P-element mobilization screen, removed the 93B6-7 to 93D3-5 cytogenetic region. [3H]uridine-labeled transcription autoradiograms revealed that normal developmental and heat shock-induced expression of the 93D puff remained unaffected in both the deficiency chromosomes. However, the amide responsivity of the 93D site was lost on the Df(3R)eP homolog while the Df(3R)eR-1 homolog responded normally to amides. Southern hybridizations with a series of upstream probes mapped the distal breakpoint of the Df(3R)eP deletion between -22 kb and -23 kb of the hsromega transcription unit. Since the distal breakpoint of Df(3R)eR-1 is at about -45 kb upstream of the hsromega gene it is inferred that the amide response element(s) that modulate the specific transcriptional activation of the 93D puff following treatment of salivary glands with a variety of amides is/are located in the -22 kb to about -45 kb upstream interval. The Df(3R)eP and Df(3R)eR-1 deletions also abolished dosage compensation at the 93D locus as well as the effect of beta-alanine levels on its heat shock inducibility.

Specific induction of the hsr omega locus of Drosophila melanogaster by amides.

We report here that 3-aminobenzamide and other amides, such as formamide, acetamide and nicotinamide, specifically induce a high rate of transcription at the 93D puff (the hsr omega heat shock gene) in polytene chromosomes of Drosophila melanogaster. Other chemicals, such as benzamide, colchicine, thiamphenicol and paracetamol, that are already known to specifically induce transcription at the hsr omega locus are also identified as amides. In view of the specific induction of the 93D puff by different amides and other data that demonstrate hsr omega transcription in response to benzamide and colchicine etc. to be independent of its heat shock induction, it appears likely that amides induce this locus through distinct regulatory elements that we propose to designate amide response elements (AREs).

Synthesis of a ubiquitously present new HSP60 family protein is enhanced by heat shock only in the Malpighian tubules of Drosophila.

A homologue of the chaperonin protein of the HSP60 family has not been shown so far in Drosophila. Using an antibody specific to HSP60 family protein in Western blotting and immunocytochemistry, we showed that a 64-kDa polypeptide, homologous to the HSP60, is constitutively present in all tissues of Drosophila melanogaster throughout the life cycle from the freshly laid egg to all embryonic, larval and adult stages. A 64-kDa polypeptide reacting with the same antibody in Western blots is present in all species of Drosophila examined. Using Western blotting in conjunction with 35S-methionine labeling of newly synthesized proteins and immuno-precipitation of the labeled proteins with HSP60-specific antibody, it was shown that synthesis of the 64-kDa homologue of HSP60 is appreciably increased by heat shock only in the Malpighian tubules, which are already known to lack the common HSPs.

The 93D (hsr-omega) locus of Drosophila: non-coding gene with house-keeping functions.

The 93D, or hsr-omega (heat-shock RNA-omega), locus of Drosophila melanogaster and other species of Drosophila, besides being induced as a member of the heat shock gene family, is also selectively and singularly inducible by a variety of agents, notably benzamide, colchicine and vitamin B6 (in species other than D. melanogaster). The genomic structure of this locus is highly conserved in all species, although the primary base sequence has diverged rapidly between species. Three transcripts (two nuclear and one cytoplasmic) are produced by this locus but none of them has any significant protein coding capacity. The profile of the three transcripts varies in a developmental and inducer-specific manner. This locus is developmentally active in nearly all cell types and is essential for viability of flies. Its induction during heat shock is independent of the other members of the heat shock gene family. The other selective inducers act on this locus through separate response elements. hsr-omega activity has a characteristic effect on transcription/turnover of the heat shock induced hsp70 and the alpha-beta transcripts in D. melanogaster. It appears that the hsr-omega locus has important house-keeping functions in transport and turnover of some transcripts and in monitoring the 'health' of the translational machinery of the cell.

RNA polymerase II dependent genes that do not code for protein.

In recent years more and more examples of RNA polymerase II dependent non-coding transcripts have been described. Although these have frequently been ignored as "selfish DNA elements", it is becoming increasingly clear that many, if not all, of them have very important biological roles. Examples of such "genes" from Drosophila, mammals, other vertebrates, yeast etc. are considered. Although the specific mechanisms through which these non-coding transcripts function in the cell are not clear, comparisons reveal certain common themes, particularly the importance of secondary structures, rather than the primary base sequence of these transcripts. While some of these transcripts may function as ribozymes or as anti-sense regulators, most others may function more directly through their specific protein-binding properties. Since RNA is believed to be the first "living" molecule, it is very likely that some genes even today function only through this class of molecules. It is expected that instead of being ignored as examples of "selfish DNA", a more positive search for their functions will help unravel the significance of this novel class of genes.