P-TEFb Regulates Transcriptional Activation in Non-coding RNA Genes

Many non-coding RNAs (ncRNAs) serve as regulatory molecules in various physiological pathways, including gene expression in mammalian cells. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. It is thus important to understand transcriptional regulation in ncRNA genes but is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA polymerase II (Pol II) promoter-proximal pausing and in a tissue-specific manner. In this study, human ncRNA genes that are expressed in the early G1 phase, termed immediate early ncRNA genes, were monitored to assess the function of positive transcription elongation factor b (P-TEFb), a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is induced in the G0–G1 transition and regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb-dependent transcription of serum responsive ncRNA genes was observed: Pol II carboxyl-terminal domain phosphorylated at serine 2 (S2) was largely increased in the transcription start site (TSS, -300 to +300) whereas overall, it was decreased in the gene body (GB, > +350) upon chemical inhibition of P-TEFb. In addition, the three representative, immediate early ncRNAs, whose expression is dependent on P-TEFb, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear enriched abundant transcript 1 (NEAT1), and X-inactive specific transcript (XIST), were further analyzed for determining P-TEFb association. Taken together, our data suggest that transcriptional activation of many human ncRNAs utilizes the pausing and releasing of Pol II, and that the regulatory mechanism of transcriptional elongation in these genes requires the function of P-TEFb. Furthermore, we propose that ncRNA and mRNA transcription are regulated by similar mechanisms while P-TEFb inhibition unexpectedly increases S2 Pol II phosphorylation in the TSSs in many ncRNA genes.One Sentence Summary: P-TEFb regulates Pol II phosphorylation for transcriptional activation in many stimulus-inducible ncRNA genes

Increased O-GlcNAcylation of Drp1 by amyloid-beta promotes mitochondrial fission and dysfunction in neuronal cells.

As a dynamic organelle, mitochondria continuously fuse and divide with adjacent mitochondria. Imbalance in mitochondria dynamics leads to their dysfunction, which implicated in neurodegenerative diseases. However, how mitochondria alteration and glucose defect contribute to pathogenesis of Alzheimer's disease (AD) is still largely unknown. Dynamin-related protein 1 (Drp1) is an essential regulator for mitochondria fission. Among various posttranslational modifications, O-GlcNAcylation plays a role as a sensor for nutrient and oxidative stress. In this study, we identified that Drp1 is regulated by O-GlcNAcylation in AD models. Treatment of Aβ as well as PugNAc resulted in mitochondrial fragmentation in neuronal cells. Moreover, we found that AD mice brain exhibits an upregulated Drp1 O-GlcNAcylation. However, depletion of OGT inhibited Drp1 O-GlcNAcylation in Aβ-treated cells. In addition, overexpression of O-GlcNAc defective Drp1 mutant (T585A and T586A) decreased Drp1 O-GlcNAcylation and Aβ-induced mitochondria fragmentation. Taken together, these finding suggest that Aβ regulates mitochondrial fission by increasing O-GlcNAcylation of Drp1

Primary cilia mediate mitochondrial stress responses to promote dopamine neuron survival in a Parkinson’s disease model

A primary cilium is an antenna-like structure on the cell surface that plays a crucial role in sensory perception and signal transduction. Mitochondria, the ‘powerhouse’ of the cell, control cell survival, and death. The cellular ability to remove dysfunctional mitochondria through mitophagy is important for cell survival. We show here that mitochondrial stress, caused by respiratory complex inhibitors and excessive fission, robustly stimulates ciliogenesis in different types of cells including neuronal cells. Mitochondrial stress-induced ciliogenesis is mediated by mitochondrial reactive oxygen species generation, subsequent activation of AMP-activated protein kinase and autophagy. Conversely, abrogation of ciliogenesis compromises mitochondrial stress-induced autophagy, leading to enhanced cell death. In mice, treatment with mitochondrial toxin, MPTP elicits ciliary elongation and autophagy in the substantia nigra dopamine neurons. Blockade of cilia formation in these neurons attenuates MPTP-induced autophagy but facilitates dopamine neuronal loss and motor disability. Our findings demonstrate the important role of primary cilia in cellular pro-survival responses during mitochondrial stress. © 2019, The Author(s).1

Peroxisome quality control and dysregulated lipid metabolism in neurodegenerative diseases

Neurodegenerative diseases: The role of peroxisome organelles Systematic studies of cellular organelles called peroxisomes are needed to determine their influence on the progression of neurodegenerative diseases. Peroxisomes play vital roles in biological processes including the metabolism of lipids and reactive oxygen species, and the synthesis of key molecules, including bile acid and cholesterol. Disruption to peroxisome activity has been linked to metabolic disorders, cancers and neurodegenerative conditions. Dong-Hyung Cho at Kyungpook National University in Daegu, South Korea, and coworkers reviewed current understanding of peroxisome regulation, with a particular focus on brain disorders. The quantity and activity of peroxisomes alter according to environmental and stress cues. The brain is lipid-rich, and even small changes in fatty acid composition may influence neuronal function. Changes in fatty acid metabolism are found in early stage Alzheimer’s and Parkinson’s diseases, but whether peroxisome disruption is responsible requires clarification

Post-Translational Modifications of ATG4B in the Regulation of Autophagy

Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A–D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy

Suppression of Cpn10 increases mitochondrial fission and dysfunction in neuroblastoma cells.

To date, several regulatory proteins involved in mitochondrial dynamics have been identified. However, the precise mechanism coordinating these complex processes remains unclear. Mitochondrial chaperones regulate mitochondrial function and structure. Chaperonin 10 (Cpn10) interacts with heat shock protein 60 (HSP60) and functions as a co-chaperone. In this study, we found that down-regulation of Cpn10 highly promoted mitochondrial fragmentation in SK-N-MC and SH-SY5Y neuroblastoma cells. Both genetic and chemical inhibition of Drp1 suppressed the mitochondrial fragmentation induced by Cpn10 reduction. Reactive oxygen species (ROS) generation in 3-NP-treated cells was markedly enhanced by Cpn10 knock down. Depletion of Cpn10 synergistically increased cell death in response to 3-NP treatment. Furthermore, inhibition of Drp1 recovered Cpn10-mediated mitochondrial dysfunction in 3-NP-treated cells. Moreover, an ROS scavenger suppressed cell death mediated by Cpn10 knockdown in 3-NP-treated cells. Taken together, these results showed that down-regulation of Cpn10 increased mitochondrial fragmentation and potentiated 3-NP-mediated mitochondrial dysfunction in neuroblastoma cells

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression

Suppression of Cpn10 exacerbates 3-NP-mediated mitochondrial dysfunction in SK-N-MC cells.

<p>(A) SK-N-MC cells were transfected with either scrambled siRNA or Cpn10 siRNA (si#1, si#2) with or without Drp1 siRNA (siDrp1) for 5 days. The cells were further exposed to 3-NP (10 mM) for 8 hr, then cells with fragmented mitochondria were counted with a fluorescence microscope. (B, C) SK-N-MC cells were transfected with either scrambled siRNA or Cpn10 siRNA (si#1, si#2) with or without Drp1 siRNA (siDrp1) for 5 days, then cells were further exposed to 3-NP (10 mM) for 8 hr. The total cellular ATP level was measured by an ATP bioluminescence detection assay (B). The intracellular ROS level was determined by a DCFH-DA fluorescence ROS detection assay (C). Data are represented as the mean ± SEM (n>3), and were considered significant at a value of *<i>p</i><0.02, **<i>p</i><0.05.</p

Down-regulation of Cpn10 promotes mitochondrial dysfunction in SK-N-MC cells.

<p>(A-C), SK-N-MC cells were transfected with a control scrambled (Sc) or Cpn10 siRNA (si #1, si #2). After 5 days, the alteration of mitochondrial membrane potential was monitored by the MitoProbe JC-1 assay (A). The cellular total ATP level was examined by an ATP bioluminescence detection assay (B). The Intracellular ROS level was measured by a DCFH-DA fluorescence ROS detection assay (C). Data are represented as the mean ± SEM (n>3), and were considered significant at a value of *<i>p</i><0.05.</p