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Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

ABSTRACT: Cells respond to stress through adaptive mechanisms that limit cellular damage and prevent cell death. MicroRNAs act as regulators of stress responses and stress can impact the functioning of miRNA biogenesis pathways. We were interested in the effect that severe proteotoxic stress capable of inducing apoptosis may have on miRNA biogenesis and the impact of the molecular chaperone protein HSP70 under these conditions. We found that the miRNA processing enzymes Drosha and Dicer and their accessory proteins DGCR8 and TRBP2 are cleaved by caspases in apoptotic cells. Overexpression of HSP70 prevented caspase activation and the degradation of these processing proteins. Caspase cleavage of TRBP2 was mapped to amino acid 234 which separates the two dsRNA-binding domains from the C-terminal Dicer interacting domain. Overexpression of TRBP2 was found to increase miRNA maturation, while expression of either of the fragments generated by caspase cleavage impaired maturation. These results indicate that inactivation of miRNA biogenesis is a critical feature of apoptosis and that cleavage of TRBP2, rather than simply a loss of function, serves to create positive acting inhibitors of pre-miRNA maturation.

SUBMITTER: Abou Zeid LY

PROVIDER: S-EPMC8821752 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It is known from earlier studies that the heat shock (HS) response in Malpighian tubules (MTs) of Drosophila larvae is different from that in other tissues because instead of the Hsp70 and other common heat shock proteins, Hsp64 and certain other new proteins are induced immediately after HS. In the present study, we examined the kinetics of the synthesis of Hsp70 and Hsp64 immediately after HS and during recovery from HS by 35S-methionine labeling and Western blotting. In addition, we also examined the transcriptional activity of hsp70 genes in larval MT cells at different times after HS by in situ hybridization and Northern blotting. The HS-induced synthesis of Hsp64 ceased by 1 hour of recovery from the HS when synthesis of the Hsp70 commenced. Our results revealed that the induced synthesis of Hsp64 immediately after HS was dependent on new transcription. Although the levels of Hsp70 in MT cells rapidly increased after its synthesis began during recovery, the levels of Hsp64 remained unaltered irrespective of its new synthesis occurring during or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the hsp70 genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3' untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the beta-galactosidase activity in 2 transgenic lines carrying the LacZ reporter gene under hsp70 promoter and different lengths of the 5'UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5'UTR.

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Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

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