• Subhash C Lakhotia

Articles written in Journal of Biosciences

• Epigenetics of heterochromatin

• Foreword

• Heat shock genes – integrating cell survival and death

Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-𝜅B mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria, formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK, NF-𝜅B and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti- and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3, essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. 𝛼𝛽 crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsr𝜔 transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins, block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as weak links” between various hubs” in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins, can act as hubs” in these networks. In view of the integrative nature of living systems, it is not surprising that stress-induced genes, generally believed to primarily function in cell survival pathways, inhibit or even promote cell death pathways at multiple levels to ensure homeostasis at cell and/or organism level. The heat shock genes obviously do much more than merely help cells survive stress.

• Pleiotropic consequences of misexpression of the developmentally active and stress-inducible non-coding hsr𝜔 gene in Drosophila

The non-coding hsr𝜔 gene of Drosophila melanogaster is expressed in nearly all cell types and developmental stages. However, in the absence of conventional mutant alleles of this gene, its developmental functions remain largely unknown. In the present study, we used a variety of GAL4 drivers to overexpress or ablate this gene’s transcripts in specific tissues and examined the developmental consequences thereof. Our results show that a balanced expression of these non-coding transcripts is critical for survival and normal development in all the tissue types tested, since any change in cellular levels of these transcripts in a given cell type generally has detrimental effects, with extreme cases resulting in organismal lethality, although in a few cases the misexpression of these transcripts also suppresses the mutant phenotype due to other genetic conditions. Evidence is also presented for existence of a new spliced variant of the hsr𝜔-n nuclear transcript. Following the RNAi-mediated down-regulation of hsr𝜔 transcripts, the omega speckles disappear so that the nucleoplasmic hnRNPs get diffusely distributed, while upregulation of these transcripts results in greater sequestration of these proteins into omega speckle clusters; either of these conditions would affect activities of the hnRNPs and other hsr𝜔-RNA interacting proteins, which is likely to have cascading consequences. The present findings, together with our earlier observations on effects of altered levels of the hsr𝜔 transcripts on induced apoptosis and expanded polyQ-mediated neurodegeneration, further confirm that ncRNA species like the hsr𝜔, far from being evolutionary hangovers, provide critical information for important functions in normal cells.

• Forty years of the 93D puff of Drosophila melanogaster

The 93D puff of Drosophila melanogaster became attractive in 1970 because of its singular inducibility by benzamide and has since then remained a major point of focus in my laboratory. Studies on this locus in my and several other laboratories during the past four decades have revealed that

this locus is developmentally active,

it is a member of the heat shock gene family but selectively inducible by amides,

the 93D or heat shock RNA omega (hsr𝜔) gene produces multiple nuclear and cytoplasmic large non-coding RNAs (hsr𝜔-n, hsr𝜔-pre-c and hsr𝜔-c),

a variety of RNA-processing proteins, especially the hnRNPs, associate with its &gt;10 kb nuclear (hsr𝜔-n) transcript to form the nucleoplasmic omega speckles,

its genomic architecture and hnRNP-binding properties with the nuclear transcript are conserved in different species although the primary base sequence has diverged rapidly,

heat shock causes the omega speckles to disappear and all the omega speckle associated proteins and the hsr𝜔-n transcript to accumulate at the 93D locus,

the hsr𝜔-n transcript directly or indirectly affects the localization/stability/activity of a variety of proteins including hnRNPs, Sxl, Hsp83, CBP, DIAP1, JNK-signalling members, proteasome constituents, lamin C, ISWI, HP1 and poly(ADP)-ribose polymerase and

a balanced level of its transcripts is essential for the orderly relocation of various proteins, including hnRNPs, RNA pol II and HP1, to developmentally active chromosome regions during recovery from heat stress. In view of such multitudes of interactions, it appears that large non-coding RNAs like those produced by the hsr𝜔 gene may function as hubs to coordinate multiple cellular networks and thus play important roles in maintenance of cellular homeostasis.

• The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster

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. Here we show 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. We suggest that besides the non-coding hsr𝜔-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsr𝜔 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.

• The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster

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. Here we show 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. We suggest that besides the non-coding hsr𝜔-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsr𝜔 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.

• # Journal of Biosciences

Current Issue
Volume 44 | Issue 2
June 2019