Over-expression of Hsp83 in grossly depleted hsrω lncRNA background causes synthetic lethality and l(2)gl phenocopy in Drosophila
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We examined interactions between the 83 kDa heat-shock protein (Hsp83) and hsrω long noncoding RNAs (lncRNAs) in hsrω66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω66 Hsp90GFP progeny died before the third instar. Rare Sp/CyO; hsrω66 Hsp90GFP reached the third instar stage but phenocopied l(2)gl mutants, becoming progressively bulbous and transparent with enlarged brain and died after prolonged larval life. Additionally, ventral ganglia too were elongated. However, hsrω66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω66/hsrω66, Sp/CyO; hsrω66/hsrω66, +/+; Hsp90GFP/Hsp90GFP and Sp/CyO; hsrω66 Hsp90GFP/hsrω66 Hsp90GFP late third instar larvae) revealed similar effects on many genes in hsrω66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerous additional genes were affected in Sp/CyO; hsrω66 Hsp90GFP larvae, with l(2)gl and several genes regulating the central nervous system being highly down-regulated in surviving Sp/CyO; hsrω66 Hsp90GFP larvae, but not in hsrω66 or Hsp90GFP single mutants. Hsp83 and several omega speckle-associated hnRNPs were bioinformatically found to potentially bind with these gene promoters and transcripts. Since Hsp83 and hnRNPs are also known to interact, elevated Hsp83 in an altered background of hnRNP distribution and dynamics, due to near absence of hsrω lncRNAs and omega speckles, can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumour-suppressor genes such as l(2)gl.
KeywordsdFUS hnRNPs kuz mmp2 SPARC TDP-43 Trithorax
We thank the Bloomington Drosophila Stock Ctr and Dr Stephen W. Mckechnie (Australia) and Dr Renato Paro (Switzerland) for providing fly stocks. We thank Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) for anti-Wingless and anti-Dlg, and Prof. Robert Tanguay (Canada) for anti-Hsp83 antibodies. We thank the Department of Biotechnology, Govt. of India (New Delhi) and the Indian Council of Medical Research (New Delhi) for supporting this research. We also thank the Centre of Advanced Studies in Department of Zoology, DBT-BHU Interdisciplinary School of Life Sciences and the Centre of Genetic Disorders (CGD) at BHU for various facilities. Special thanks to Dr Amit Chaurasia of Premas Biotech, CGD, for RNA-sequencing. We acknowledge the Department of Science and Technology, Govt. of India (New Delhi) and the Banaras Hindu University for Confocal Microscopy facility. We thank Dr Yashvant Patel in our lab for help in RNA sequence analysis. This work was supported by a CEIB-II grant from the Department of Biotechnology, Govt. of India (no. BT/PR6150/COE/34/20/2013) to SCL. MR was supported as a senior research fellow by the Indian Council of Medical Research, New Delhi, India.
The NGS data for RNA-sequencing in the first set of genotypes (Oregon R+ (WT), Sp/CyO; hsrω66, Sp/CyO; Hsp90GFP, Sp/CyO; hsrω66 Hsp90GF) are deposited at the GEO (http://www.ncbi.nlm.nih.gov/geo/) with accession no. GSE116476. RNA-seq data, while accession no. for the second set of genotypes (WT and +/+; hsrω66) is GSE120077.
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