Publications by authors named "Yulia V Surovtseva"

25 Publications

  • Page 1 of 1

Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription.

Mol Biol Cell 2021 Apr 10;32(9):956-973. Epub 2021 Mar 10.

Department of Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, CT 06520.

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions (NORs), and there are 10 in a human diploid genome. While there are 10 NORs, however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is two to three per cell. Here, to identify novel regulators of ribosome biogenesis we used high-throughput quantitative imaging of MCF10A cells to identify proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this unique screening approach led to identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through the S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression.
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http://dx.doi.org/10.1091/mbc.E20-10-0670DOI Listing
April 2021

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro.

bioRxiv 2020 Oct 21. Epub 2020 Oct 21.

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed -1 ribosomal frameshifting (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting -1 PRF as an effective antiviral strategy for SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.10.21.349225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587830PMC
October 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Screening Novel Agent Combinations to Expedite CTCL Therapeutic Development.

J Invest Dermatol 2021 Jan 12;141(1):217-221. Epub 2020 Jun 12.

Department of Dermatology, Yale School of Medicine, New Haven, Connecticut, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2020.05.097DOI Listing
January 2021

JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.

Blood Adv 2020 05;4(10):2213-2226

Department of Dermatology, Yale School of Medicine, New Haven, CT.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing T lymphocytes that is more likely to involve the peripheral blood in advanced stages. For such patients with advanced disease, there are few available systemic treatment options, and prognosis remains poor. Exome sequencing studies of CTCL have suggested therapeutic targets, including within the JAK/STAT pathway, but JAK inhibition strategies may be limited by patient-specific mutational status. Because our recent research has highlighted the potential roles of single and combination approaches specifically using BCL2, bromodomain and extra-terminal domain (BET), and histone deacetylase (HDAC) inhibition, we aimed to investigate the effects of JAK inhibition on CTCL cells and established CTCL cell lines when paired with these and other targeting agents. Peripheral blood malignant CTCL isolates exhibited differential responses to JAK inhibition, with JAK2 expression levels negatively correlating to 50% inhibitory concentration (IC50) values. Regardless of single-agent sensitivity, JAK inhibition potentiated malignant cell cytotoxicity in combination with BCL2, BET, HDAC, or proteasome inhibition. Combination inhibition of JAK and BCL2 showed the strongest potentiation of CTCL cytotoxicity, driven by both intrinsic and extrinsic apoptosis pathways. JAK inhibition decreased expression of BCL2 in the high-responder samples, suggesting a putative mechanism for this combination activity. These results indicate that JAK inhibition may have major effects on CTCL cells, and that combination strategies using JAK inhibition may allow for more generalized cytotoxic effects against the malignant cells from patients with CTCL. Such preclinical assessments help inform prioritization for combination targeted drug approaches for clinical utilization in the treatment of CTCL.
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http://dx.doi.org/10.1182/bloodadvances.2020001756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252559PMC
May 2020

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

J Biol Chem 2019 08 21;294(32):12146-12156. Epub 2019 Jun 21.

Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut 06520. Electronic address:

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity are not suitable for high-throughput drug screening. The finding that the PkPSD from can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC values ranging from 3.1 to 42.3 μm Lead compounds were evaluated against the pathogenic yeast in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native PSD mitochondrial activity and growth, with an MIC of 22.5 and 15 μg/ml without ethanolamine and an MIC of 75 and 60 μg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.
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http://dx.doi.org/10.1074/jbc.RA119.007766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690695PMC
August 2019

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

ACS Chem Biol 2018 12 20;13(12):3286-3293. Epub 2018 Nov 20.

Department of Microbial Pathogenesis , Yale School of Medicine , New Haven , Connecticut 06536 , United States.

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.
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http://dx.doi.org/10.1021/acschembio.8b00714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001666PMC
December 2018

PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling.

Proc Natl Acad Sci U S A 2018 09 4;115(38):E8996-E9005. Epub 2018 Sep 4.

Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520;

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.
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http://dx.doi.org/10.1073/pnas.1804379115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156646PMC
September 2018

Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number.

Cell Rep 2018 02;22(7):1923-1934

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address:

Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2-3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.
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http://dx.doi.org/10.1016/j.celrep.2018.01.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828527PMC
February 2018

A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis.

J Biol Chem 2018 02 15;293(5):1493-1503. Epub 2017 Dec 15.

From the Basic Science Section, Department of Medicine, National Jewish Health, Denver, Colorado 80206,

Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with PSD (MBP-His-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λ = 403 nm, λ = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.
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http://dx.doi.org/10.1074/jbc.RA117.000525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798280PMC
February 2018

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM.

Proc Natl Acad Sci U S A 2017 05 12;114(21):5503-5508. Epub 2017 May 12.

Department of Neurosurgery, Yale School of Medicine, New Haven, CT 06520;

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the -bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.
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http://dx.doi.org/10.1073/pnas.1702942114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448170PMC
May 2017

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

Sci Transl Med 2017 02;9(375)

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.
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http://dx.doi.org/10.1126/scitranslmed.aal2463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435119PMC
February 2017

Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay.

J Am Chem Soc 2016 Mar 9;138(11):3844-55. Epub 2016 Mar 9.

Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.

Small-molecule inhibitors of DNA repair pathways are being intensively investigated as primary and adjuvant chemotherapies. We report the discovery that cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, are potent inhibitors of DNA double-strand break (DSB) repair. Our data suggest that cardiac glycosides interact with phosphorylated mediator of DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin-protein ligase ring finger protein 8 (RNF8), two factors involved in DSB repair, and inhibit the retention of p53 binding protein 1 (53BP1) at the site of DSBs. These observations provide an explanation for the anticancer activity of this class of compounds, which has remained poorly understood for decades, and provide guidance for their clinical applications. This discovery was enabled by the development of the first high-throughput unbiased cellular assay to identify new small-molecule inhibitors of DSB repair. Our assay is based on the fully automated, time-resolved quantification of phospho-SER139-H2AX (γH2AX) and 53BP1 foci, two factors involved in the DNA damage response network, in cells treated with small molecules and ionizing radiation (IR). This primary assay is supplemented by robust secondary assays that establish lead compound potencies and provide further insights into their mechanisms of action. Although the cardiac glycosides were identified in an evaluation of 2366 small molecules, the assay is envisioned to be adaptable to larger compound libraries. The assay is shown to be compatible with small-molecule DNA cleaving agents, such as bleomycin, neocarzinostatin chromophore, and lomaiviticin A, in place of IR.
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http://dx.doi.org/10.1021/jacs.6b00162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530877PMC
March 2016

Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

J Biol Chem 2016 Jan 19;291(2):989-97. Epub 2015 Nov 19.

From the Departments of Pathology and Genetics, Yale School of Medicine, New Haven, Connecticut 06520-8023,

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.
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http://dx.doi.org/10.1074/jbc.M115.689299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705416PMC
January 2016

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+ NIK+ signalosome on Rab5+ endosomes.

Proc Natl Acad Sci U S A 2015 Aug 20;112(31):9686-91. Epub 2015 Jul 20.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; Department of Pathology, Yale University School of Medicine, New Haven, CT 06520

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.
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http://dx.doi.org/10.1073/pnas.1503535112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534258PMC
August 2015

Transcription-independent role for human mitochondrial RNA polymerase in mitochondrial ribosome biogenesis.

Nucleic Acids Res 2013 Feb 8;41(4):2479-88. Epub 2013 Jan 8.

Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.

Human mitochondrial RNA polymerase, POLRMT, is required for mitochondrial DNA (mtDNA) transcription and forms initiation complexes with human mitochondrial transcription factor B2 (h-mtTFB2). However, POLRMT also interacts with the paralogue of h-mtTFB2, h-mtTFB1, which is a 12S ribosomal RNA methyltransferase required for small (28S) mitochondrial ribosome subunit assembly. Herein, we show that POLRMT associates with h-mtTFB1 in 28S mitochondrial ribosome complexes that are stable in the absence of mitochondrial transcription and distinct from transcription complexes containing POLRMT and h-mtTFB2. Overexpression of POLRMT in HeLa cells increases 12S rRNA methylation by h-mtTFB1 and reduces the steady-state levels of mtDNA-encoded proteins and respiration, apparently because of a decrease in fully assembled 55S mitochondrial ribosomes. We propose that POLRMT interacts directly with h-mtTFB1 in 28S mitochondrial ribosomes to augment its 12S rRNA methyltransferase activity, and that together they provide a checkpoint for proper 28S and 55S mitochondrial ribosome assembly. Thus, POLRMT is multi-functional, forming distinct protein complexes that regulate different steps in mitochondrial gene expression, at least one of which does not involve transcription per se. The significance of these results is discussed with regard to the mechanism and regulation of human mitochondrial gene expression and the potential multi-functionality of RNA polymerases in general.
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http://dx.doi.org/10.1093/nar/gks1447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575816PMC
February 2013

Mitochondrial ribosomal protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription.

Proc Natl Acad Sci U S A 2011 Nov 14;108(44):17921-6. Epub 2011 Oct 14.

Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.

Basal transcription of human mitochondrial DNA (mtDNA) in vitro requires the single-subunit, bacteriophage-related RNA polymerase, POLRMT, and transcription factor h-mtTFB2. This two-component system is activated differentially at mtDNA promoters by human mitochondrial transcription factor A (h-mtTFA). Mitochondrial ribosomal protein L7/L12 (MRPL12) binds directly to POLRMT, but whether it does so in the context of the ribosome or as a "free" protein in the matrix is unknown. Furthermore, existing evidence that MRPL12 activates mitochondrial transcription derives from overexpression studies in cultured cells and transcription experiments using crude mitochondrial lysates, precluding direct effects of MRPL12 on transcription to be assigned. Here, we report that depletion of MRPL12 from HeLa cells by shRNA results in decreased steady-state levels of mitochondrial transcripts, which are not accounted for by changes in RNA stability. We also show that a significant "free" pool of MRPL12 exists in human mitochondria not associated with ribosomes. "Free" MRPL12 binds selectively to POLRMT in vivo in a complex distinct from those containing h-mtTFB2. Finally, using a fully recombinant mitochondrial transcription system, we demonstrate that MRPL12 stimulates promoter-dependent and promoter-independent transcription directly in vitro. Based on these results, we propose that, when not associated with ribosomes, MRPL12 has a second function in transcription, perhaps acting to facilitate the transition from initiation to elongation. We speculate that this is one mechanism to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, where transcription of rRNAs from the mtDNA presumably needs to be adjusted in accordance with the rate of import and assembly of the nucleus-encoded MRPs into ribosomes.
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http://dx.doi.org/10.1073/pnas.1108852108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207702PMC
November 2011

LRP130 protein remodels mitochondria and stimulates fatty acid oxidation.

J Biol Chem 2011 Dec 4;286(48):41253-41264. Epub 2011 Oct 4.

Division of Cardiovascular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605. Electronic address:

Impaired oxidative phosphorylation (OXPHOS) is implicated in several metabolic disorders. Even though mitochondrial DNA encodes several subunits critical for OXPHOS, the metabolic consequence of activating mitochondrial transcription remains unclear. We show here that LRP130, a protein involved in Leigh syndrome, increases hepatic β-fatty acid oxidation. Using convergent genetic and biochemical approaches, we demonstrate LRP130 complexes with the mitochondrial RNA polymerase to activate mitochondrial transcription. Activation of mitochondrial transcription is associated with increased OXPHOS activity, increased supercomplexes, and denser cristae, independent of mitochondrial biogenesis. Consistent with increased oxidative phosphorylation, ATP levels are increased in both cells and mouse liver, whereas coupled respiration is increased in cells. We propose activation of mitochondrial transcription remodels mitochondria and enhances oxidative metabolism.
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http://dx.doi.org/10.1074/jbc.M111.276121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308838PMC
December 2011

Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes.

Mol Cell 2009 Oct;36(2):207-18

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.

Orthologs of the yeast telomere protein Stn1 are present in plants, but other components of the Cdc13/Stn1/Ten1 (CST) complex have only been found in fungi. Here we report the identification of conserved telomere maintenance component 1 (CTC1) in plants and vertebrates. CTC1 encodes an approximately 140 kDa telomere-associated protein predicted to contain multiple OB-fold domains. Arabidopsis mutants null for CTC1 display a severe telomere deprotection phenotype accompanied by a rapid onset of developmental defects and sterility. Telomeric and subtelomeric tracts are dramatically eroded, and chromosome ends exhibit increased G overhangs, recombination, and end-to-end fusions. AtCTC1 both physically and genetically interacts with AtSTN1. Depletion of human CTC1 by RNAi triggers a DNA damage response, chromatin bridges, increased G overhangs, and sporadic telomere loss. These data indicate that CTC1 participates in telomere maintenance in diverse species and that a CST-like complex is required for telomere integrity in multicellular organisms.
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http://dx.doi.org/10.1016/j.molcel.2009.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768651PMC
October 2009

STN1 protects chromosome ends in Arabidopsis thaliana.

Proc Natl Acad Sci U S A 2008 Dec 8;105(50):19815-20. Epub 2008 Dec 8.

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.

Telomeres shield the natural ends of chromosomes from nucleolytic attack, recognition as double-strand breaks, and inappropriate processing by DNA repair machinery. The trimeric Stn1/Ten1/Cdc13 complex is critical for chromosome end protection in Saccharomyces cerevisiae, while vertebrate telomeres are protected by shelterin, a complex of six proteins that does not include STN1 or TEN1. Recent studies demonstrate that Stn1 and Ten1 orthologs in Schizosaccharomyces pombe contribute to telomere integrity in a complex that is distinct from the shelterin components, Pot1 and Tpp1. Thus, chromosome-end protection may be mediated by distinct subcomplexes of telomere proteins. Here we report the identification of a STN1 gene in Arabidopsis that is essential for chromosome-end protection. AtSTN1 encodes an 18-kDa protein bearing a single oligonucleotide/oligosaccharide binding fold with significant sequence similarity to the yeast Stn1 proteins. Plants null for AtSTN1 display an immediate onset of growth and developmental defects and reduced fertility. These outward phenotypes are accompanied by catastrophic loss of telomeric and subtelomeric DNA, high levels of end-to-end chromosome fusions, increased G-overhang signals, and elevated telomere recombination. Thus, AtSTN1 is a crucial component of the protective telomere cap in Arabidopsis, and likely in other multicellular eukaryotes.
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http://dx.doi.org/10.1073/pnas.0807867105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2604966PMC
December 2008

Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance.

EMBO J 2007 Aug 12;26(15):3653-61. Epub 2007 Jul 12.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.

POT1 is a single-copy gene in yeast and humans that encodes a single-strand telomere binding protein required for chromosome end protection and telomere length regulation. In contrast, Arabidopsis harbors multiple, divergent POT-like genes that bear signature N-terminal OB-fold motifs, but otherwise share limited sequence similarity. Here, we report that plants null for AtPOT1 show no telomere deprotection phenotype, but rather exhibit progressive loss of telomeric DNA. Genetic analysis indicates that AtPOT1 acts in the same pathway as telomerase. In vitro levels of telomerase activity in pot1 mutants are significantly reduced and are more variable than wild-type. Consistent with this observation, AtPOT1 physically associates with active telomerase particles. Although low levels of AtPOT1 can be detected at telomeres in unsynchronized cells and in cells arrested in G2, AtPOT1 binding is significantly enhanced during S-phase, when telomerase is thought to act at telomeres. Our findings indicate that AtPOT1 is a novel accessory factor for telomerase required for positive telomere length regulation, and they underscore the coordinate and extraordinarily rapid evolution of telomere proteins and the telomerase enzyme.
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http://dx.doi.org/10.1038/sj.emboj.7601792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949013PMC
August 2007

The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection.

Mol Cell Biol 2005 Sep;25(17):7725-33

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.

Pot1 (protection of telomeres 1) is a single-stranded telomere binding protein that is essential for chromosome end protection and telomere length homeostasis. Arabidopsis encodes two Pot1-like proteins, dubbed AtPot1 and AtPot2. Here we show that telomeres in transgenic plants expressing a truncated AtPot1 allele lacking the N-terminal oligonucleotide/oligosaccharide binding fold (P1DeltaN) are 1 to 1.5 kb shorter than in the wild type, suggesting that AtPot1 contributes to the positive regulation of telomere length control. In contrast, telomere length is unperturbed in plants expressing the analogous region of AtPot2. A strikingly different phenotype is observed in plants overexpressing the AtPot2 N terminus (P2DeltaC) but not the corresponding region in AtPot1. Although bulk telomeres in P2DeltaC mutants are 1 to 2 kb shorter than in the wild type, these plants resemble late-generation telomerase-deficient mutants with severe growth defects, sterility, and massive genome instability, including bridged chromosomes and aneuploidy. The genome instability associated with P2DeltaC mutants implies that AtPot2 contributes to chromosome end protection. Thus, Arabidopsis has evolved two Pot genes that function differently in telomere biology. These findings provide unanticipated information about the evolution of single-stranded telomere binding proteins.
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http://dx.doi.org/10.1128/MCB.25.17.7725-7733.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190295PMC
September 2005

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis.

J Biol Chem 2004 Nov 10;279(46):47799-807. Epub 2004 Sep 10.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.
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http://dx.doi.org/10.1074/jbc.M407938200DOI Listing
November 2004

A new enrichment approach identifies genes that alter cell cycle progression in Saccharomyces cerevisiae.

Curr Genet 2004 Jun 12;45(6):350-9. Epub 2004 Mar 12.

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, USA.

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.
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http://dx.doi.org/10.1007/s00294-004-0497-5DOI Listing
June 2004