The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. This study investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster. Pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. Deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR (Funikov, 2016).

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. This study provides the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. This study identified Drosophila CG11321, which was named Linear Ubiquitin E3 ligase (LUBEL), and it was found to catalyze linear ubiquitination in vitro. Endogenous linear ubiquitin chain-derived peptides were detected by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, linear ubiquitination-defective flies were established by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies (Asaoka, 2016).

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. It was predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, basal levels of gene expression were compared at different developmental temperatures within each of the two species. Furthermore, the heat hardening dynamics were explored by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster an indication was found of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively (Sorensen, 2019).

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, 2012), 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. 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. 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 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. 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. 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).

Previous work has demonstrated that species of the Stratiomyidae family exhibit higher tolerance to thermal stress in comparison with that of many representatives of Diptera, including Drosophila species. It was hypothesized that species of this group inherited the specific structures of their chaperones from an ancestor of the Stratiomyidae family, and this enabled the descendants to colonize various extreme habitats. To explore this possibility, copies of the Hsp70 genes from Stratiomys singularior, a typical eurythermal species, and Drosophila melanogaster, were cloned and expressed in Escherichia coli for comparison. To investigate the thermal sensitivity of the chaperone function of the inducible 70-kDa heat shock proteins from these species, an in vitro refolding luciferase assay was used. Under conditions of elevated temperature, S. singularior Hsp70 exhibited higher reactivation activity in comparison with D. melanogaster Hsp70 and even human Hsp70. Similarly, S. singularior Hsp70 was significantly more thermostable and showed in vitro refolding activity after preheatment at higher temperatures than D. melanogaster paralog. Thermally induced unfolding experiments using differential scanning calorimetry indicated that Hsp70 from both Diptera species is formed by two domains with different thermal stabilities and that the ATP-binding domain of S. singularior is stable at temperatures 4 degrees higher than that of the D. melanogaster paralog. This study represents the first report that provides direct experimental data indicating that the evolutionary history of a species may result in adaptive changes in the structures of chaperones to enable them to elicit protective functions at extreme environments (Garbuz, 2019).

Environmental changes cause stress, Reactive Oxygen Species and unfolded protein accumulation which hamper synaptic activity and trigger cell death. Heat shock proteins (HSPs) assist protein refolding to maintain proteostasis and cellular integrity. Mechanisms regulating the activity of HSPs include transcription factors and posttranslational modifications that ensure a rapid response. HSPs preserve synaptic function in the nervous system upon environmental insults or pathological factors and contribute to the coupling between environmental cues and neuron control of development. A biased screening was conducted in Drosophila melanogaster searching for synaptogenic modulators among HSPs during development. The role of two small-HSPs (sHSPs). sHSP23 and sHSP26 was explored in synaptogenesis and neuronal activity. Both sHSPs immunoprecipitate together and the equilibrium between both chaperones is required for neuronal development and activity. The molecular mechanism controlling HSP23 and HSP26 accumulation in neurons relies on a novel gene (CG1561). which was named Pinkman (pkm). It is proposed that sHSPs and Pkm are targets to modulate the impact of stress in neurons and to prevent synapse loss (Santana, 2020).

Hsp67Bc in Drosophila melanogaster is a member of the small heat shock protein family, the main function of which is to prevent the aggregation of misfolded or damaged proteins. Hsp67Bc interacts with Starvin and Hsp23, which are known to be a part of the cold-stress response in the fly during the recovery phase. This study investigated the role of the Hsp67Bc gene in the cold-stress response. In adult Drosophila, Hsp67Bc expression was shown to increase after cold stress and decrease after 1.5 h of recovery, indicating the involvement of Hsp67Bc in short-term stress recovery. A deletion in the D. melanogaster Hsp67Bc gene was implemented using imprecise excision of a P-element, and the cold tolerance of Hsp67Bc-null mutants was analyzed at different developmental stages. Hsp67Bc-null homozygous flies were found to be viable and fertile but display varying cold-stress tolerance throughout the stages of ontogenesis: the survival after cold stress is slightly impaired in late 3(rd) instar larvae, unaffected in pupae, and notably affected in adult females. Moreover, the recovery from chill coma is delayed in Hsp67Bc-null adults of both sexes. In addition, the deletion in the Hsp67Bc gene caused more prominent up-regulation of Hsp70 following cold stress, suggesting the involvement of Hsp70 in compensation of the lack of the Hsp67Bc protein. Taken together, these results suggest that Hsp67Bc is involved in the recovery of flies from a comatose state and contributes to the protection of the fruit fly from cold stress (Malkeyeva, 2021).

His4r is the only known variant of histone H4 in Drosophila. It is encoded by the His4r single-copy gene that is located outside of the histone gene cluster and expressed in a different pattern than H4, although the encoded polypeptides are identical. A null mutant (His4r(Δ42)) was generated which is homozygous viable and fertile without any apparent morphological defects. Heterozygous His4r(Δ42) is a mild suppressor of position-effect variegation, suggesting that His4r has a role in the formation or maintenance of condensed chromatin. Under standard conditions loss of His4r has a modest effect on gene expression. Upon heat-stress the induction of the Heat shock protein (HSP) genes Hsp27 and Hsp68 is stronger in His4r(Δ42) mutants with concordantly increased survival rate. Analysis of chromatin accessibility after heat shock at a Hsp27 regulatory region showed less condensed chromatin in the absence of His4r while there was no difference at the gene body. Interestingly, preconditioning before heat shock led to increased chromatin accessibility, HSP gene transcription and survival rate in control flies while it did not cause notable changes in His4r(Δ42). Thus, these results suggest that His4r might play a role in fine tuning chromatin structure at inducible gene promoters upon environmental stress conditions (Farago, 2021).

With the rapid development of science and technology, 5G technology will be widely used, and biosafety concerns about the effects of 5G radiofrequency radiation on health have been raised. Drosophila melanogaster was selected as the model organism for this study, in which a 3.5 GHz radiofrequency radiation (RF-EMR) environment was simulated at intensities of 0.1 W/m(2), 1 W/m(2), and 10 W/m(2). The activity of parent male and offspring (F1) male flies was measured using a Drosophila activity monitoring system under short-term and long-term 3.5 GHz RF-EMR exposure. Core genes associated with heat stress, the circadian clock and neurotransmitters were detected by QRT-PCR technology, and the contents of GABA and glutamate were detected by UPLC-MS. The results show that short-term RF-EMR exposure increased the activity level and reduced the sleep duration while long-term RF-EMR exposure reduced the activity level and increased the sleep duration of F1 male flies. Under long-term RF-EMR, the expression of heat stress response-related hsp22, hsp26 and hsp70 genes was increased, the expression of circadian clock-related per, cyc, clk, cry, and tim genes was altered, the content of GABA and glutamate was reduced, and the expression levels of synthesis, transport and receptor genes were altered. In conclusion, long-term RF-EMR exposure enhances the heat stress response of offspring flies and then affects the expression of circadian clock and neurotransmitter genes, which leads to decreased activity, prolonged sleep duration, and improved sleep quality (Wang, 2021).

Under exposure to harmful environmental stresses, organisms exhibit a general stress response involving upregulation of the expression of heat shock proteins (HSPs) which is thought to be adaptive. Small heat shock proteins (sHSPs) are key components of this response, although shsp genes may have other essential roles in development. However, the upregulation of expression of a suite of genes under stress may not necessarily be evidence of an adaptive response to stress that involves those genes. To explore this issue, the CRISPR/Cas9 system was used to investigate pleiotropic effects of the hsp23 gene in Drosophila melanogaster. Transgenic flies carrying a pCFD5 plasmid containing sgRNAs were created to generate a complete knockout of the hsp23 gene. The transgenic line lacking hsp23 showed an increased hatch rate and no major fitness costs under an intermediate temperature used for culturing the flies. In addition, hsp23 knockout affected tolerance to hot and cold temperature extremes but in opposing directions; knockout flies had reduced tolerance to cold, but increased tolerance to heat. Despite this, hsp23 expression (in wild type flies) was increased under both hot and cold conditions. The hsp23 gene was required for heat hardening at the pupal stage, but not at the 1st-instar larval stage, even though the gene was upregulated in wild type controls at that life stage. The phenotypic effects of hsp23 were not compensated for by expression changes in other shsps. This study shows that the fitness consequences of an hsp gene knockout depends on environmental conditions, with potential fitness benefits of gene loss even under conditions when the gene is normally upregulated (Gu, 2021).

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. This study finds that the m(6)A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m(6)A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, this study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m(6)A RNA methylation and demonstrates their roles in suppressing autophagy (Tang, 2021).

mTORC1, an evolutionarily conserved serine/threonine kinase, is a master regulator of cell growth, metabolism, and proliferation coupling different nutritional and environmental cues, including growth factors, energy levels, cellular stress, and amino acids, with metabolic programs. For example, insulin activates PI3K/AKT and inhibits the Tuberous Sclerosis Complex (TSC) 1/2, a negative regulator of mTORC1, thus promoting mTORC1 activation. Activated mTORC1 then phosphorylates multiple downstream effectors that control a wide range of anabolic and catabolic processes. Phosphorylation of the ribosomal S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1) by mTORC1 promotes protein translation and enhances cell growth and proliferation. Moreover, autophagy, an intracellular degradation system that delivers cytoplasmic components to lysosomes, is inhibited by mTORC1 through phosphorylation of Atg13 that, in turn, inhibits ULK1 kinase activity (Tang, 2021).

Recent studies have highlighted a role for mTORC1 in regulating RNA metabolism. Through the phosphorylation of RNA metabolic proteins, mTORC1 modulates various RNA biogenesis and processing events. Phosphorylation of the SR protein kinase SRPK2 by S6K1 promotes its transport into the nucleus where it activates SR proteins and induces splicing of lipogenic pre-messenger RNAs (pre-mRNAs) for de novo synthesis of fatty acids and cholesterol, suggesting that SRPK2 is a critical mediator of mTORC1-dependent lipogenesis. In addition, mTORC1 regulates alternative splicing and polyadenylation of autophagic and metabolic genes to control autophagy, lipid, protein, and energy metabolism through the cleavage and polyadenylation complex. Furthermore, mTORC1 mediates phosphorylation of the decapping enzyme Dcp2. Phosphorylated Dcp2 associates with RNA helicase RCK family members and binds to transcripts of Autophagy-related genes (Atg) to degrade them, thereby suppressing autophagy. Altogether, these studies suggest an essential role for mTORC1 in controlling RNA biogenesis and processing, revealing a major function for mTORC1 in the regulation of protein diversity and in reshaping cellular metabolism and autophagy (Tang, 2021).

N6-methyl-adenosine (m6A) is one of the most abundant chemical modifications in eukaryotic mRNA, which is preferentially enriched in 3' UTRs and around stop codons. m6A modification affects almost all aspects of mRNA metabolism, such as splicing, translation, and stability, and plays essential roles in a wide range of cellular processes, including Drosophila sex determination and metabolism. The m6A methyltransferase complex (MTC) catalyzes m6A formation and is composed of the methyltransferase-like protein 3 (METTL3), the methyltransferase-like protein 14 (METTL14), WTAP (the ortholog of Drosophila Fl(2)d), and RBM15/RBM15B (the ortholog of Drosophila Nito). Although METTL3 is the only catalytic component of the MTC, its interaction with METTL14 is necessary for RNA substrate recognition and efficient m6A deposition. WTAP stabilizes the interaction between the two METTL proteins, and RBM15/RBM15B have been proposed to recruit the MTC to its target transcripts (Tang, 2021).

Using autophagy as a readout of mTORC1 signaling in Drosophila, the MTC was identified as a downstream effector of mTORC1 signaling. From the analysis of high-confidence Drosophila and human MTC proteomic data, the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex was identified as an MTC interactor that mediates the effects of mTORC1 on m6A modification and autophagy. In mammalian cells, it was also found that the CCT complex plays critical roles in the regulation of MTC protein stability and m6A RNA modification, suggesting that the mTORC1-CCT-MTC axis is conserved from Drosophila to mammals. These studies thus unveil a mechanism linking mTORC1 signaling and the chaperonin CCT complex to RNA methylation and also uncover a layer of mTORC1 regulation of autophagy (Tang, 2021).

This study has demonstrate that the MTC acts as a downstream effector of mTORC1 to regulate m6A RNA methylation of Atg transcripts, inducing their degradation and thus suppressing autophagy. Furthermore, the CCT complex was identified as a link between mTORC1 and MTC. CCT downstream of mTORC1 signaling can stabilize METTL3 and METTL14 to up-regulate m6A levels and inhibit autophagy. Accordingly, depletion of either mTORC1, CCT, METTL3, or METTL14 compromises m6A RNA methylation and promotes autophagy. Importantly, the role of mTORC1-CCT-MTC signaling in regulating autophagy is conserved from Drosophila to mammals. Thus, this study discovered a function of mTORC1 in regulating m6A RNA methylation during autophagy (Tang, 2021).

mTORC1 inhibition suppresses protein translation but also affects gene expression at different levels. This study identified an epitranscriptomic mechanism by which mTORC1 activates m6A RNA methylation to promote Atg mRNA turnover and inhibits autophagy. This m6A-mediated mRNA degradation represents a layer of gene regulation by mTORC1. Moreover, as mTORC1 activity regulates global m6A levels, it is likely that the MTC also mediates additional physiological functions of mTORC1. It is noted that depletion of METTL3, METTL14, or CCT8 cannot fully rescue TSC1-induced effects. Although these results could be caused by partial RNAi knockdown, they may also indicate that other pathways contribute to mTORC1 regulation of autophagy. Indeed, studies have reported that mTORC1 suppresses autophagy through modulation of transcription factors, RNA-processing complexes, and mRNA degradation machinery, further highlighting that mTORC1 utilizes multiple RNA biogenesis processes to control autophagy (Tang, 2021).

The catalytic core components of the MTC, METTL3/METTL14, have a substrate sequence specificity for a DRA*CH motif (D = G/A/U, R = G/A, A* = methylated adenosine, H = A/U/C). However, only a subset of consensus sites across the mRNA transcriptome are methylated. Thus, it has been speculated that other factors in the MTC specify METTL3/METTL14 methylation patterns. Proteomic results combined with biochemical validation in both Drosophila and mammalian cells identified multiple splicing factors that interact with known MTC components. Future work will be needed to confirm whether these factors are directly involved in the regulation of m6A methylation and how they coordinate with the m6A machinery to affect RNA processing. It will also be interesting to investigate whether mTORC1 controls other regulators of RNA m6A methylation, in addition to METTL3 and METTL14. Moreover, the proteomics data revealed that multiple components of E3 ubiquitin ligase complex interact with MTC, suggesting that they may be involved in ubiquitination of MTC. Ubiquitination of METTL3 has also been observed, but its function and related E3 ubiquitin ligases remain unclear (Tang, 2021).

Previous genetic analyses showed that the CCT complex functions downstream of mTORC1 and that mTORC1 positively regulates the transcriptional levels of the CCT complex (Kim, 2019). Another study identified CCT2 as a substrate of S6 kinase, a downstream effector of mTOR, in mammalian cells, suggesting that both transcriptional and posttranslational regulations contribute to CCT complex activation by mTORC1. However, the phosphorylation site (Ser-260) of mammalian CCT2 is not conserved in Drosophila and how this phosphorylation modulates CCT function is not clear. Multiple phosphorylation sites have been detected in CCT components. Interestingly, a previous study showed that CCT8 was phosphorylated following insulin stimulation (Vinayagam, 2016), suggesting that other phosphorylation sites are involved in mTORC1-regulated CCT activation. Future studies are needed to comprehensively map the phosphorylation sites on CCT components and investigate their physiological roles (Tang, 2021).

The CCT complex is a highly conserved complex that assists the folding of about 10% of the eukaryotic proteome. The interactions of the CCT complex with METTL3 and METTL14 were observed in a previous study using AP/MS in human cells. Consistently, genetic and biochemical data from this study further confirmed their interactions and characterized the functions of CCT in stabilizing METTL3 and METTL14 and controlling m6A RNA methylation. These findings thus further expand the impact of the CCT complex on RNA metabolism (Tang, 2021).

Multiple studies have reported that CCT complex protein levels dramatically increase in autophagy mutants, proposing that CCT is one of the substrates of autophagy. Future studies will be needed to test whether autophagy is able to degrade the CCT complex and whether autophagy feedback inhibits CCT (Tang, 2021).

Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing aggregation of misfolded proteins, it is involved in macroautophagy regulation alongside the Starvin protein. Overexpression of the D. melanogaster Hsp67Bc gene has been shown to stimulate macroautophagy in S2 cell culture. Nonetheless, it has been unknown how the absence of the Hsp67Bc gene may affect it. The effect of Hsp67Bc gene deletion was studied on the macroautophagy induced by the pathogenic Wolbachia wMelPop strain in D. melanogaster. Wolbachia was detected inside autophagic vacuoles in fly neurons, thereby proving that these endosymbionts were being eliminated via macroautophagy. Nevertheless, no difference was registered in brain bacterial load between Hsp67Bc-null and control flies at all tested stages of ontogenesis. Moreover, the abundance of autophagic vacuoles was similar between neurons of the mutant and control flies, yet the cross-sectional area of autolysosomes on ultrathin sections was more than 1.5-fold larger in Hsp67Bc-null fly brains than in the control line. These findings suggest that the product of the Hsp67Bc gene does not participate in the initiation of endosymbiont-induced macroautophagy but may mediate autophagosome maturation: the deletion of the Hsp67Bc gene leads to the increase in autolysosome size (Malkeyeva, 2021).

Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Public scRNA-seq datasets from Drosophila melanogaster midgut cells were used in this study to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication (Silva, 2021).

Plastic responses to heat stress have been shown to temporarily increase heat stress tolerance in many small ectotherms. Heat shock proteins (Hsps) have previously been shown to play a role in this induced heat stress tolerance. The heat shock response is fast but short lived, with the cellular Hsp concentration peaking within a few hours after induction. Induced heat stress tolerance, on the other hand, peaks 16-32 hours after induction. Therefore, the inducible heat stress response must depend on additional mechanisms. The Turandot gene family has been suggested as a candidate. It contains eight genes that are all upregulated to some degree following heat stress in Drosophila melanogaster. Previously, Turandot A (totA) and Turandot X (totX) have been linked to induced heat stress tolerance. This study aimed to investigate the temporal dynamics of Turandot expression and the functional role of totA and totC for heat stress tolerance. This was done by assaying the temporal heat tolerance and Turandot gene expression after a heat insult, and by exposing Turandot gene knock down flies to a range of heat hardening treatments, and evaluating the effects on heat tolerance. Successful gene knock down was verified by gene expression assays. In addition, expression of hsp70A was included. Both totA, totC, and hsp70A expression increased following a heat hardening treatment, while the results for totX were less clear. The expression of totC temporally co-occurred with and was functionally linked to increased heat tolerance. Expression of totA did not have a significant effect on heat stress tolerance. The complexity of inducible heat tolerance was underlined by the result that knock down of Turandot genes led to increased expression of hsp70. The results suggest that heat tolerance is determined by the interaction between several mechanisms, of which Turandot genes constitute one such mechanism (Amstrup Bay, 2022).

Cancer cells generally overexpress heat shock proteins (Hsps), the major components of cellular stress response, to overcome and survive the diverse stresses. However, the specific roles of Hsps in initiation and establishment of cancers remain unclear. Using loss of Lgl-mediated epithelial tumorigenesis in Drosophila, tumorigenic somatic clones of different genetic backgrounds were induced to examine the temporal and spatial expression and roles of major heat shock proteins in tumor growth. The constitutively expressed Hsp83, Hsc70 (heat shock cognate), Hsp60 and Hsp27 show elevated levels in all cells of the tumorigenic clone from early stages that persists until their transformation. However, the stress-inducible Hsp70 is expressed only in a few cells at later stage of established tumorous clones that show high F-actin aggregation. Intriguingly, levels of Heat shock factor (HSF), the master regulator of Hsps, remain unaltered in these tumorous cells and its down-regulation does not affect tumorigenic growth of lgl^- clones overexpressing Yorkie, although down-regulation of Hsp83 prevents their survival and growth. Interestingly, overexpression of HSF or Hsp83 in lgl^- cells makes them competitively successful in establishing tumorous clones. These results show that the major constitutively expressed Hsps, but not the stress-inducible Hsp70, are involved in early as well as late stages of epithelial tumors and their elevated expression in lgl^- clones co-overexpressing Yorkie is independent of HSF (Singh, 2022).

Benzene, a ubiquitous environmental chemical, is known to cause immune dysfunction and developmental defects. This study aims to investigate the relation between benzene-induced immune dysfunction and developmental toxicity in a genetically tractable animal model, Drosophila melanogaster. Further, the study explored the protective role of Heat Shock Protein 70 (Hsp70) against benzene-induced immunotoxicity and subsequent developmental impact. Drosophila larvae exposed to benzene (1.0, 10.0, and 100.0 mM) were examined for total hemocyte (immune cells) count, phagocytic activity, oxidative stress, apoptosis, and their developmental delay and reduction were analyzed. Benzene exposure for 48 h reduced the total hemocytes count and phagocytic activity, along with an increase in the Reactive Oxygen Species (ROS), and lipid peroxidation in the larval hemocytes. Subsequently, JNK-dependent activation of the apoptosis (Caspase-3 dependent) was also observed. During their development, benzene exposure to Drosophila larvae led to 3 days of delay in development, and ~40% reduced adult emergence. Hsp70-overexpression in hemocytes was found to mitigate benzene-induced oxidative stress and abrogated the JNK-mediated apoptosis in hemocytes, thus restoring total hemocyte count and improving phagocytotic activity. Further, hsp70-overexpression in hemocytes also lessened the benzene-induced developmental delay (rescue of 2.5 days) and improved adult emergence (~20%) emergence, revealing a possible control of immune cells on the organism's development and survival. Overall, this study established that hsp70-overexpression in the Drosophila hemocytes confers protection against benzene-induced immune injury via regulating the ROS/JNK signaling pathway, which helps in the organism's survival and development (D'Souza, 2022).

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Search PubMed for articles about Drosophila Heat shock factor

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