In apoptosis, mitochondrial outer membrane permeabilization (MOMP) triggers caspase-dependent death. However, cells undergo clonogenic death even if caspases are blocked. One proposed mechanism involved the release of cytotoxic proteins (e.g., AIF and endoG) from mitochondria. To initiate MOMP directly without side effects, we created a tamoxifen-switchable BimS fusion protein. Surprisingly, even after MOMP, caspase-inhibited cells replicated DNA and divided for approximately 48 h before undergoing proliferation arrest. AIF and endoG remained in mitochondria. However, cells gradually lost mitochondrial membrane potential and ATP content, and DNA synthesis slowed to a halt by 72 h. These defects resulted from a partial loss of respiratory function, occurring 4-8 h after MOMP, that was not merely due to dispersion of cytochrome c. In particular, Complex I activity was completely lost, and Complex IV activity was reduced by approximately 70%, whereas Complex II was unaffected. Later, cells exhibited a more profound loss of mitochondrial protein constituents. Thus, under caspase inhibition, MOMP-induced clonogenic death results from a progressive loss of mitochondrial function, rather than the release of cytotoxic proteins from mitochondria.

The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.

The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

The processing and metabolism of amyloid precursor protein (APP) is a major interest in Alzheimer disease (AD) research, because not only amyloid beta (Abeta) peptide, but also cellular or mitochondrial APP are intimately involved in cellular dysfunction and AD pathogenesis. Here we demonstrate that APP is directly and efficiently cleaved by the HtrA2 serine protease in vitro and in vivo. Using several APP mutants and N-terminal amino acid sequencing, we identified that the HtrA2-mediated APP cleavage product is the C161 fragment encompassing amino acids 535-695 of APP695. The immunofluorescence and subcellular fractionation studies indicate that APP is partly colocalized with HtrA2 in the mitochondria where HtrA2 can cleave APP under normal conditions. The HtrA2-cleaved C161 fragment was detected in the cytosolic fraction; therefore, we postulate that the C161 fragment is released into the cytosol after cleavage of APP by HtrA2. Interestingly, the level of C161 was remarkably decreased in motor neuron degeneration (mnd2) mice in which the serine protease activity of HtrA2 was greatly reduced. These results show that the protease activity of HtrA2 is essential for the production of C161 and that processing of APP into C161 is a natural event occurring under normal physiological conditions. Our study suggests that the direct cleavage of mitochondrial APP by HtrA2 may prevent mitochondrial dysfunction caused by accumulation of APP and that the regulation of HtrA2 protease activity may be a therapeutic target in AD.