Dimer-specific immunoprecipitation of active caspase-2 identifies TRAF proteins as novel activators.
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ABSTRACT:
SUBMITTER: Robeson AC
PROVIDER: S-EPMC6043850 | biostudies-literature | 2018 Jul
REPOSITORIES: biostudies-literature
Publications
Dimer-specific immunoprecipitation of active caspase-2 identifies TRAF proteins as novel activators.
Robeson Alexander C AC Lindblom Kelly R KR Wojton Jeffrey J Kornbluth Sally S Matsuura Kenkyo K
The EMBO journal 20180606 14
Caspase-2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase-2 bimolecular fluorescence complementation (BiFC) system with fluorophore-specific immunoprecipitation to isolate and study the active caspase-2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor-associated ...[more]
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Project description:Study questionCan novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary?Summary answerSYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis.What is known alreadyThe phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown.Study design size, durationWe carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates).Participants/materials, setting, methodsUsing quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3' untranslated region (3'UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B.Main results and the role of chanceWe identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity.Large scale dataThe raw data are available at GEO using the study ID: GSE81524.Limitations, reasons for cautionThis analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets.Wider implications of the findingsThese data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility.Study funding and competing interest(s)This study was supported by the UK Medical Research Council (grant no G1100357 to R.A.A. and an intramural MRC programme grant to I.R.A.). The authors declare no competing interests.
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