bluuebunny/crossref_metadata_2025 huggingface dataset:
Palladin Functionally Replaces the Arp2/3 Complex and Organizes Actin‐Rich Structures Generated during Listeria monocytogenes Infections
Authors: Dhanda Arandeep S. , Vogl A Wayne , Otey Carol A. , Beck Moriah R. , Guttman Julian A.
Published: June 2021

Listeria monocytogenes (Listeria) co‐opts the actin cytoskeleton of host cells for their entry, intracellular motility and dissemination. These bacteria generate Arp2/3‐based, branched actin‐containing structures called comet tails that propel the bacteria within and amongst host cells. The host protein palladin is a crucial component of actin‐rich structures. Whether this protein can promote actin based motility events remains unclear. Here we tested the hypothesis that palladin is a crucial organizer of actin structures generated during Listeria infections and simultaneously used Listeria as a model to establish if palladin could compensate for processes requiring the Arp2/3 complex. Using immunofluorescence microscopy we identified palladin at bacterial invasion sites and motile comet tails. Strikingly, depletion of palladin via RNAi results in shorter and severely misshapen comet tails. When palladin mutants defective for actin or VASP binding are expressed in cells, comet tails disintegrate or become progressively thinner as they move. Through transmission electron microscopy, we demonstrate that these thin comet tails result from a switch in the actin network from branched arrays to parallel F‐actin bundles. To determine whether palladin could compensate for the Arp2/3 complex during comet tail motility, we overexpressed palladin in cells treated with the potent Arp2/3 inhibitor CK‐666. In these cells, Listeria can initiate and maintain its actin‐based motility. Additionally, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells Listeria fail to generate comet tails. However, when palladin is overexpressed in this Arp2/3 functionally null cell line, the ability of Listeria to generate actin clouds and comet tails is restored. Taken together, we show that palladin is essential for the organization of F‐actin structures generated by Listeria and, importantly, can compensate for Arp2/3 complex functional defects without hindrance during bacterial actin‐based motility.

Support or Funding Information

Grant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397 to A.W.V), NIH (grant no. R15 GM120670 to M.R.B) and, SFU departmental funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tau does not protect microtubules during Klebsiella pneumoniae infections
Authors: Bogdan Alexander Constantine , Chua Michael Dominic , Guttman Julian Andrew
Published: June 2021

Hospitals have raised a growing concern for the rampant spread of the bacterial pathogen Klebsiella pneumoniae, which can cause a wide array of symptoms such as pneumonia, urinary tract infections, septicemia, and pyogenic liver abscesses. Moreover, the misuse of antibiotics has contributed to the surge of multidrug‐resistant hypervirulent strains. As a result, therapeutics for K. pneumoniae infections are becoming ineffective leading to lower survival rates and higher mortality rates in highly infected individuals. In our laboratory, we have shown that K. pneumoniae activates katanin microtubule severing enzymes to induce microtubule disassembly in lung epithelial cells. In contrast, microtubule associated proteins (MAPs) in host cells regulate the stability of microtubules and specific MAPs such as Tau (MAPTau) can protect microtubules from katanin‐induced severing. Since Tau plays a role in the stability of microtubules, we hypothesized that Tau protein levels may prevent microtubule disassembly during K. pneumoniae infections. To test this hypothesis, we initially determined if K. pneumoniae targets Tau in infected A549 lung epithelial cells. We lysed uninfected and infected cells and we found that a higher molecular weight Tau species was present in the infected cell lysates. Hyperphosphorylation of Tau typically regulates its binding to microtubules, and thus, we then determined if overexpressing Tau constructs that have different phosphorylation abilities could protect microtubules from K. pneumoniae‐induced katanin‐mediated severing. To do this, we obtained three green fluorescent protein (EGFP)‐tagged tau constructs: wildtype Tau (wt Tau), hyperphosphorylated Tau (E14 Tau), and Tau that cannot be phosphorylated (AP Tau). We then expressed these plasmids in A549 cells and infected the cells with K. pneumoniae. If Tau protects microtubules from katanin‐severing, E14 Tau should have no effect on K. pneumoniae‐induced severing while wild‐type Tau and AP Tau should prevent microtubule severing. During these infections no rescue of the microtubule phenotype was observed thus, neither wild‐type Tau nor AP Tau protected microtubules from K. pneumoniae‐triggered microtubule disassembly. Our findings show that Tau overexpression plays no discernible role in protecting microtubules during Klebsiella pneumoniae infections.

Support or Funding Information

This study was funded by SFU institutional funds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of novel actin‐associated proteins at enteropathogenic Escherichia coli and Listeria monocytogenes actin‐rich structures
Authors: Chua Michael Dominic , Walker Brittany Dominique , Li Serina , Guttman Julian Andrew
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes generate actin‐rich structures that are used for their colonization of host cells. EPEC remains extracellular, attaches to the host plasma membrane and secretes a variety of effector proteins that are used to control the host cells. The most dramatic morphological phenotype generated during these infections is the formation of actin‐rich protrusions called pedestals that are needed for E. coli‐based disease. L. monocytogenes enters its host cell and once in the cytosol it generates a bacterial protein (called ActA) that recruits actin polymerizing proteins to 1 pole of the microbe forming a comet tail that is used for movement within and amongst the host cells Previously, our lab conducted a mass spectrometry analysis of concentrated EPEC pedestals and over 90 novel proteins were identified. From this list, we selected a subset for confirmatory analysis and to determine whether their presence required the pre‐formation of the actin‐rich structures. These proteins included a calponin protein (CNN), dihydropyrimidinase‐like protein (CRMP4), nucleoside kinase (NK), mitogen‐activated protein pathway kinase (MK), and a ubiquitin‐conjugating enzyme (Ube2N). Because these novel proteins were observed in EPEC pedestals, we hypothesized that these five protein candidates are important for EPEC pedestals and L. monocytogenes comet tails. To study this, we first confirmed that these proteins were enriched in EPEC pedestals by immunolocalizing the proteins in EPEC‐infected cells. CNN, and NK were found throughout the full length of the pedestal while CRMP4, MK, and Ube2N were concentrated at the apical tip of EPEC pedestals. In L. monocytogenes‐infected cells, CNN and NK were found in actin clouds, comet tails, and listeriopods. CRMP4 immunolocalized only at invasion sites and listeriopods while MK was only present at listeriopods. To ensure that recruitment of these proteins were independent of bacterial attachment on the host cell and required formation of the actin‐rich structures, we infected cells with either a tir‐deficient EPEC mutant or an actA‐deficient L. monocytogenes mutant as both mutants cannot generate actin‐structures. None of the five proteins were recruited to the mutants suggesting that their presences are dependent on the formation of the actin‐rich structures. From these results, we have identified novel proteins that are important for the various stages of EPEC and L. monocytogenes infections. By identifying these proteins, we have begun to elucidate the complex web of proteins involved in the formation and maintenance of actin structures during these infections.

Support or Funding Information

This study was funded through NSERC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The highly conserved proteins Nucleolin and Sub1 play critical roles in regulating G4 DNA‐induced genome instability.
Authors: Kim Nayun
Published: June 2021

Sequences containing multiple guanine‐runs can form the four‐stranded G‐quadruplex (G4) DNA. The significance of G4 DNA in etiology of blood cancers is underscored by the manifest enrichment of G4 sequence motifs proximal to the leukemia/lymphoma‐associated translocation breakpoints. But how G4 DNA‐motifs instigate chromosome abnormalities leading to malignant transformation is yet to be resolved. We developed quantitative assays to measure and characterize multiple types of G4 DNA‐induced genome instability events in the context of the yeast genome. This system allowed us to demonstrate that the level and direction of transcription are important determinants of the stability of G4 sequences. Using this approach, we demonstrated that the topological change ensuing in the absence of Topoisomerase I (Top1) can severely endanger genome instability at actively transcribed G4 motifs. The G4‐associated instability in top1Δ yeasts was suppressed partially by the bacterial topA (ecTopA), which removes negative supercoils and not at all by the gyrases (ecGyrase), which removes positive supercoils or the RNase H1 overexpression, which removes RNA:DNA hybrids or R‐loops. Taking advantage of the G4 DNA‐conducive cellular environment in the top1Δ yeasts, we also demonstrated that, upon disruption of Sub1, a general transcription co‐activator, genome instability linked to co‐transcriptionally formed G4 DNA is significantly elevated and that the highly conserved DNA binding domain of Sub1 and its human homolog PC4 is sufficient in preventing genome instability at actively transcribed G4 motifs. Consistent with its role in suppressing G4‐mediated instability, Sub1 interacts specifically with co‐transcriptionally formed G4 DNA in vivo and yeast cells become highly sensitivity to G4‐stabilizing chemical ligands by the loss of Sub1. We demonstrated that Sub1 forms a complex with the G4‐resolving helicase Pif1 suggesting a possible mechanism by which Sub1 suppresses G4‐associated genome instability. Additionally, we discuss our most recent findings regarding the role of the highly conserved nucleolar protein Nucleolin (Nsr1 in yeast) in G4 DNA‐associated genome instability. Deletion of Nsr1, a known G4 DNA‐binding protein, surprisingly led to a significant decrease of G4‐induced recombination. We present a model where interaction with Nsr1 shift the equilibrium between B DNA and G4 DNA toward stable G4 DNA and thereby create strong genome‐destabilizing replication block.

Support or Funding Information

Work in the NK lab was supported by grants from the Welch Foundation (AU1875) and the National Institutes of Health (GM116007).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NELF‐E and CDYL1: Two novel players for switching off transcription at DNA damage sites
Authors: Ayoub Nabieh , Awwad Samah W. , Abu‐Zhayia Enas R. , Ben‐Oz Bella M. , Guttmann‐Raviv Noga Guttmann‐Raviv
Published: June 2021

Transcription activity is rapidly and transiently paused in response to DNA double‐strand breaks (DSBs) to eliminate production of abnormal transcripts and to avoid deleterious collisions between transcription and repair machineries. Little is known about the mechanisms that ensure transcription repression following DNA damage. Here we describe two novel DNA‐damage responsive proteins, regulated by PARP1 activity, that foster DSB‐induced transcription silencing and promote DSB repair. First, using I‐SceI and CRISPR‐Cas9 endonucleases, we showed that the negative elongation factor member E (NELF‐E), subunit of the NELF complex, is preferentially recruited to DSBs induced upstream transcriptionally active rather than inactive genes. The preferential recruitment of NELF‐E to DNA breakage sites is dependent on the presence of RNA polymerase II [1]. Second, we showed that the chromodomain Y‐like (CDYL1) protein is rapidly recruited to damaged euchromatic regions. In addition, CDYL1 promotes the recruitment of EZH2 methyltransferase, stimulates local increase of the repressive methyl mark H3K27me3 and promotes transcription silencing at DSB sites. Furthermore, the “traffic‐light reporter” system revealed that CDYL1 mainly promotes homology‐directed repair of DSBs. Consequently, CDYL1 knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin [2]. In the meeting, I will also discuss unpublished data implicating CDYL1 in regulating histone crotonylation at DNA damage sites. Altogether, our findings identify NELF‐E and CDYL1 as novel components that play an essential role in the cellular response to DNA damage.

Support or Funding Information

This work was supported by grants from the Israel Science Foundation (ISF) (grant no. 2021242), the Israel Cancer association (grant no. 2019404), the Binational Science Foundation (grant no. 2023065), the Israel Cancer Research Fund (ICRF) (grant no. 2021762), Volkswagen Foundation (grant no. 2020594). Enas R. Abu‐Zhaiya and Samah W. Awwad are supported by the Council for Higher Education 19 fellowship for outstanding minority MSc and PhD students, respectively. N. Ayoub is supported by the Neubauer Family Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanding the role of FDO1 in yeast chromosomal biology
Authors: Seman Melissa , Hoggard Timothy , Fox Catherine
Published: June 2021

The yeast transcription factor Forkhead 1 (FKH1) has multiple roles in chromosomal biology. Our lab recently discovered a protein‐protein interaction between Fkh1 and Fdo1 that contributes to mating type switching, one of multiple nuclear roles for Fkh1. Little information exists for roles of Fdo1 in yeast, and we were interested in whether Fdo1 has additional roles in chromosomal biology. To this end, we have begun to characterize the yeast cell cycle in a fdo1‐dependent manner. Using flow cytometry, we have uncovered connections between fdo1 and cell cycle progression as well as genome stability.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Role of the Human CST Complex in Regulating the DNA Damage Response
Authors: Stewart Jason A. , Ackerson Stephanie M.
Published: June 2021

CST (CTC1‐STN1‐TEN1) is an RPA‐like, ssDNA binding protein that functions in telomere replication and genome‐wide DNA replication rescue. Previous studies have shown that depletion of STN1 or CST overexpression leads to decreased or increased new origin firing following replication stress, suggesting a role for CST in dormant origin activation. Additionally, depletion of CST subunits leads to increased anaphase bridges and micronuclei. Recently, a conditional CTC1 knockout line was created in HCT116 cells. Removal of CTC1 was found to cause decreased cell proliferation and elongation of the telomeric ssDNA G‐overhang, leading to RPA accumulation and telomeric DNA damage signaling. Interestingly, rescue of the telomeric damage did not restore normal cell growth, suggesting that other factors contribute to the growth defects in the absence of CTC1. Here, we investigate the consequences of CTC1 deletion on genome‐wide replication and activation of the DNA damage response. We find that CTC1 deletion lead to a delayed but abrupt activation of p53 and p21 leading to decreased DNA replication and a G2/M arrest. We hypothesized that this cell cycle arrest was caused by ATR activation from defects in telomere or genome‐wide DNA replication. Instead, our results suggest that ATR signaling is defective in the absence of CTC1 and p53 is activated in an ATR‐independent manner. Overall, our findings suggest a novel and unexpected role of CST in promoting checkpoint signaling to prevent genome instability.

Support or Funding Information

This work is supported by the National Institutes of Health grant R00 GM104409 and startup funds from the University of South Carolina.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histone Deacetylase Inhibition Leads to Dose‐Dependent Suppression of Oncogene‐Associated Super‐Enhancers
Authors: Wheeler Graycen , Sanchez Gilson , Liu Xuedong
Published: June 2021

In cancer cells, decreased histone acetylation leads to gene expression patterns that drive oncogenic processes. Histone deacetylase (HDAC) inhibitors are therefore used as therapeutics to selectively kill cancer cells. The complex processes by which these chromatin‐remodeling agents affect gene expression and ultimately cell fate are still not entirely understood. The Liu group seeks to characterize the mechanisms of the HDAC inhibitor largazole by using epigenomic and transcriptomic studies to compare its effects on transformed and non‐transformed cells at different doses. From broad chromatin immunoprecipitation sequencing and RNA sequencing, we observed that largazole has a striking effect on super‐enhancer regions and the genes associated with them. Largazole decreases the accumulation of RNA polymerase II at super‐enhancer elements and limits the transcription of genes under the regulation of those super‐enhancers. Largazole also alters the expression of many genes by decreasing the histone acetylation of poised distal enhancers in a dose‐dependent manner. Many of these genes are transcriptionally upregulated after treatment, as is canonically associated with an increased histone acetylation profile, but some transcripts are repressed. These insights into the epigenetic reprogramming that drives oncogenic processes provide fundamental information about tumorigenesis and could potentially serve as a foundation for the development of further oncology therapies.

Support or Funding Information

National Institutes of Health [T32GM008759 to G.W.]; National Cancer Institute and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health [CA107098, AR068254 to X.L.]; National Cancer Institute [CA107098S1 to G.S.];

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Binding Kinetics of CTCF in vitro
Authors: Sanborn Adrian L. , Kornberg Roger D.
Published: June 2021

CTCF is a ubiquitously expressed transcription factor whose architectural role as an insulator is tied to its ability to form genomic loops and delimit boundaries of topologically associated domains (TADs). This capacity for binding of a single protein to affect distal regulation over megabases is both remarkable and poorly understood mechanistically. In current models, a cohesin‐associated complex expands a loop by tracking along DNA in both directions, while CTCF defines the loop size and position by halting this complex. However, a recent single molecule imaging study finds the DNA‐association lifetime of CTCF to be ten‐fold shorter than that of cohesin in vivo, suggesting that CTCF and cohesin act independently and dynamically (Hansen et al., 2017). This raises key questions about how CTCF forms and maintains domain boundaries and loops, and highlights the need for a careful study of CTCF‐DNA binding kinetics.

Here, we purified human CTCF and characterized its DNA‐binding activity in vitro. Using mobility shift assays, we recapitulated the known high affinity of full length CTCF and the zinc finger domain for CTCF binding sites and its dependence on DNA methylation. We further used surface plasmon resonance to measure real time binding and unbinding kinetics of CTCF to methylated and unmethylated DNA targets. These experiments illuminate the role of DNA methylation in CTCF binding site selection, shed light on in vivo binding kinetics, and represent a first step towards the study of CTCF‐mediated looping in vitro.

Support or Funding Information

This research was supported by NIH Grants GM36659 and AI21144 (to R.D.K.) and a National Defense Science and Engineering Fellowship (to A.L.S.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐based design of Nucleosome Binding Peptides for controlling cell function
Authors: Teles Kaian Amorim , Santos Guilherme Martins
Published: June 2021

Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. A myriad of histone modifications, which strongly induce changes in chromatin structure, have been associated with malignancies and other diseases, highlighting the crucial role of chromatin architectural changes in disease mechanisms. The nucleosome surface was hypothesised as a therapeutic target, however the development, design and function of exogenous nucleosome binding molecules remain unclear.

Herein, we performed structure‐based calculations on the nucleosome surface interaction to four peptides (NBMs), which present different nucleosome binding sites, derived from Nucleosome Binding Proteins (NBPs) to analyse the nucleosome sensitivity. Further, we were able to generate a new peptide (GMIP) with two nucleosome binding sites. A series of in vitro studies were performed to verify the impact of all these peptides on the nucleosome, showing differential action on its stability. We also demonstrated that GMIP and a canonical peptide may induce chromatin condensation in vitro. Confocal microscopy using labeled GMIP showed that it penetrates, by a passive mechanism, the cell and binds to chromatin. Cell‐based assays (MTT and FACS) showed that NBMs tested here presented a strong inhibitory activity on tumor cells, but not on non‐tumoral cells, proliferation and viability. It was also shown that these peptides might induce chromatin condensation in vivo. Besides that, the fish embryo toxicity (FET) test showed specificity in actions of NBMs on the ability to induce tissue modifications.

Finally, we correlated the structural impact of NBMs to cellular function, providing a unique map on the nucleosome for designing new molecules with important implications. Taking all of this into account, it is clear that the is right to consider the nucleosome surface as a potential therapeutic target to regulate a plethora of cellular disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Chimeric Histone Methyltransferase Complexes In Saccharomyces cerevisiae Generate Unique Phenotypes and Clarify the Roles of MLL1 and Set1 Complex Accessory Proteins
Authors: Chosed Renee J. , Klein David , Longan Emery , Baker Marian , Gogoli Sasha , Wang Jingtian , Alkoutami Sami
Published: June 2021

Genomic rearrangements involving the MLL1 gene are prevalent in human cancers, especially mixed lineage leukemia. This gene encodes an enzyme that methylates histone H3 on lysine 4 (H3K4) as a part of the MLL1 multi‐protein complex. The MLL1 complex regulates the expression of many genes via this epigenetic mark, including homeobox genes. Problematically, higher eukaryotes contain many functionally redundant complexes that complicate the study of MLL1 and associated cancers in a living system. Furthermore, due to the nature of the disease‐causing mutations, namely chromosomal rearrangements, MLL1 is not a promising drug target. However, several accessory proteins within the complex are required for catalytic activity, presenting possible drug targets themselves. Herein we present an in vivo system for the study of the MLL1 complex in Saccharomyces cerevisiae, making use of the homologous Set1/COMPASS complex. We genetically replaced COMPASS members from S. cerevisiae with their human homologs using antibiotic resistance cassettes. We then performed phenotypic characterization of chimeric COMPASS/MLL1 complexes assessing global H3K4 methylation status. Selected chimeric yeast‐human methyltransferase complexes conferred catalytic activity at varying degrees, while others did not confer methyltransferase activity. Notably, we observed H3K4 dimethylation levels comparable to wild type when human Ash2L replaced yeast Bre2 but reduced levels of H3K4 trimethylation with this same chimeric complex. Together, these data represent a proof of concept for simplifying the study of this clinically important protein complex in a tractable in vivo system, and also offer mechanistic insight into the functional role of a catalytically essential accessory protein within the MLL1 complex.

Support or Funding Information

This research was supported by grants from the National Center for Research Resources (5 P20 RR016461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative analysis of lncRNA from human FF and FFPE brain speimens
Authors: Lv Yehui , Rao Yuliang , Dong Wenzhe
Published: June 2021

Both Fresh frozen (FF) and formalin‐fixed paraffin‐embedded (FFPE) brain tissues are important resources for molecular medical study. However, long non‐coding RNA (lncRNA) analysis is restricted in FFPE samples due to its high‐level degradation. To compare the quality of lncRNA extracted from FF and FFPE brain specimens and to explore the possibility of lncRNA quantification in FFPE tissues, 10 paired of frozen and FFPE human brain tissues were collected from autopsies by our collaborating teams at the Institute of Criminal Scientific Technology in Shanghai (). We selected 2 endogenous control mRNA (ACTB and GAPDH) and 6 target lncRNA (MALAT1, H19, PTENP1, BC1, HAR1 and DD3) from literatures, and designed 2 short (<100 bp, non‐overlapping) and 1 long (about 200 bp) primers for each biomarker. After total RNA was extracted from all specimens, the expression level of each biomarker was analyzed by real‐time quantitative PCR (RT‐qPCR) and amplification efficiency (AE) was subsequently calculated. The degradation profiles of total RNA were evaluated by RNA integrity number (RIN), and RIN values of FFPE samples were significantly lower than FF samples (P<0.05). Following RT‐qPCR, short amplicon of all 6 RNA markers achieved optimal AE between 90% and 110% in FF samples, while AE of long amplicons differed remarkably with RNA type, and only 2 lncRNA (MALAT1 and BC1) and endogenous control mRNA reached the optimal efficiency. As for FFPE tissues, most short amplicons (except for HAR1) achieved the optimum efficiencies, however, AE of most long amplicons was overblown in FFPE samples, including ACTB and GAPDH. In present study, high concordance of RNA expression was also observed in 2 non‐overlapping short amplicons of both FF and FFPE samples. In conclusion, short amplicons of target lncRNA were amplified more efficiently than long amplicons and this study may provide an experimental basis for accurate lncRNA quantitative analysis with short amplicons in FFPE brain tissues.

Support or Funding Information

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical permission was granted through the Science and Ethics Committee of Shanghai University of Medicine & Health Sciences. This study was funded by the Seed Fund of Shanghai University of Medicine & Health Sciences and Open Foundation of Shanghai Key Laboratory of Forensic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The function of noncoding RNA BC200 in a human disease
Authors: shin heegwon , lee jungmin , kim youngmi , lee younghoon
Published: June 2021

Although BC200 RNA was originally identified as a neuron‐specific non‐coding RNA, it is overexpressed in various cancer tissues. Recently, BC200 RNA was shown to promote metastasis in several cancer cell lines, but its mechanism was not understood in detail. To determine this mechanism, we knocked down BC200 RNA in a cervical cancer cell line, HeLa, which overexpress the RNA, and examined cell motility, profiling of ribosome footprints, and the correlation between cell motility changes and genes exhibiting altered ribosome profiles. We found that knockdown of BC200 RNA decreased cell motility and altered more than 2‐fold expressions of 29 genes including S100A11 (which showed a reduced ribosome footprint) previously shown to increase cell motility. Knockdown of BC200 RNA significantly destabilized S100A11 mRNA, suggesting that the down‐regulation of S100A11 was mainly caused by loss of stability of its mRNA. Together, our results show that BC200 RNA increases cell motility by stabilizing S100A11 transcripts.

Support or Funding Information

This study was supported by National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (2017R1A6A3A11031308), and the KAIST High Risk High Return Project (HRHRP).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microarray Profiling and Bioinformatic Analysis of Circular RNAs in RAW264.7 Macrophages Under Simulated Microgravity
Authors: Yuan Ming , Wang Jingyu , Yuan Min
Published: June 2021

Macrophages play a vital role in the innate immune system. Lots of studies have showed that spaceflight can change the function of macrophage and induce the immune disorder, however, the mechanisms remain to be illustrated. circular RNAs (circRNAs) can act as miRNA sponges and play an important role in regulating gene expression, here we tested the hypothesis that simulated microgravity can induce the differential expression of circRNAs in RAW 264.7 macrophages under simulated microgravity. Arraystar Mouse circRNA Array (8x15K, Arraystar) was used to identify the circRNA expression profiles in RAW 264.7 macrophages from Control(Con) and simulated microgravity(SM) groups, the cells in SM group were treated by clinostat for 72 hours. Total RNA was quantified using the NanoDrop ND‐1000. The sample preparation and microarray hybridization were performed based on the Arraystar's standard protocols, Agilent Feature Extraction software was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering with a threshold of fold change ≥1.5 and P<0.05. Quantitative RT‐PCR was performed in the Real‐time PCR System (Applied Biosystems) using SYBR Green qPCR master mix(Arraystar). Bioinformatic analyses including gene ontology (GO) and KEGG pathway were applied to predict the potential functions of circRNAs. The results from circRNA microarrays revealed that there were 60 differentially expressed circRNAs in SM group compared with Con group, in which 34 circRNAs were found to be up‐regulated and 26 circRNAs were down‐regulated. Furthermore, the up‐regulated expression of circR003780, circR015248 and circR015947 were verified by qRT‐PCR. GO analysis indicated that the top20 up‐regulated enriched GO terms included biological process “positive regulation of chronic inflammatory response”. KEGG analysis showed that the top 30 pathway enrichment included Wnt and mTOR signaling pathway, protein processing in endoplasmic reticulum, apoptosis, et al. These results indicated that simulated microgravity can change the circRNAs expression pattern in RAW 264.7 macrophages, the differentially‐expressed circRNAs may participate in the function changes of macrophage in spaceflight.

Support or Funding Information

Funded by National Basic Research Programs of China, No.2014CB744404

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping RNA‐chromatin interactions
Authors: Zhong Sheng , Sridhar Bharat , Rivas‐Astroza Marcelo , Nguyen Tri C. , Chen Weizhong , Yan Zhangming , Cao Xiaoyi , Hebert Lucie
Published: June 2021

RNA molecules can attach to chromatin. It remains difficult to know what RNAs are associated with chromatin and where the genomic target loci of these RNAs are. Here, we present MARGI (mapping RNA‐genome interactions), a technology to massively reveal native RNA‐chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin‐associated RNAs (caRNAs) with their target genomic sequences by proximity ligation, forming RNA‐DNA chimeric sequences, which are converted to a sequencing library for paired‐end sequencing. Using MARGI, we produced RNA‐genome interaction maps for human embryonic stem cells (ESCs) and human embryonic kidney (HEK) cells. MARGI revealed hundreds of caRNAs, including previously known XIST, NEAT1, and MALAT1, as well as each caRNA's genomic interaction loci. Using a cross‐species experiment, we estimated that approximately 2.2% of MARGI‐identified interactions were false positives. In ESCs and HEK cells, the RNA ends of more than 5% of MARGI read pairs were mapped to distal or inter‐chromosomal locations as compared to the locations of their corresponding DNA ends. The majority of transcription start sites are associated with distal or inter‐chromosomal caRNAs. Chromatin‐immunoprecipitation‐sequencing (ChIP‐seq)‐reported H3K27ac and H3K4me3 levels are positively correlated, while H3K9me3 is negatively correlated, with MARGI‐reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and their genomic target regions.

Support or Funding Information

NIH DP1HD087990

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐135a Regulates NHE9 To Inhibit Proliferation and Migration of Glioblastoma Cells
Authors: Zubieta Daniela Maria Gomez , Hamood Mohamed A. , Beydoun Rami A. , Pall Ashley E. , Kondapalli Kalyan A.
Published: June 2021

Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified.

We show that microRNA 135a (miR‐135a) targets NHE9 to downregulate its expression in U87 cells. MiR‐135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR‐135a reduced proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR‐135a takes a two‐pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR‐135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR‐135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane. We propose downregulation of miR‐135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR‐135a as a potential therapeutic for glioblastomas with NHE9 overexpression.

Support or Funding Information

Funds from the Department of Natural Sciences and the Office of Research and Sponsored Programs at the University of Michigan Dearborn

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Critical RNA Structures Involved in Translation Initiation Mechanism between 3′ CITE and 5′ UTR of RNA2 of Blackcurrant Reversion Nepovirus (BRV)
Authors: Bean Scott‐Wesley M. , Baquero Galvis Laura D. , Morrison Evan J. , Filbin‐Wong Megan E.
Published: June 2021

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail of a single mRNA molecule, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Beyond this, both RNAs are unique in sequence, and they likely utilize a different translation initiation mechanism. RNA structures within a specific site of the 3′ UTRs of RNA1 and RNA2 called the cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with the 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but the exact mechanism is not well understood. It is not known whether the 3′ CITE interaction with the 5′ UTR in translation initiation is in cis or trans. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to determine which of these structures are necessary for the success of its translation initiation. Through the use of an RNA reporter construct that consists of the luciferase gene flanked by the 3′ CITE and 5′ UTR of RNA2, as well as both in vitro RNA probing and in silico modeling of folded RNA2, structures involved in translation initiation have been pinpointed. Generation of mutants which disrupted specific stem and loop structures of the RNA2 3′ CITE resulted in changes in the quantity of luciferase protein made, subsequently indicating critical RNA structures needed for translation. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.

Support or Funding Information

MSU Denver Applied Learning Center Undergraduate Research Grant, Colorado‐Wyoming Alliance for Minority Participation Undergraduate Research Grant, MSU Denver Dean's Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Angiotensin II Induced Long Non‐coding RNA GIVER Regulates Oxidative Stress, Inflammation, and Proliferation in Vascular Smooth Muscle Cells
Authors: Zhang Erli , Das Sadhan , Amaram Vishnu , Reddy Marpadga A. , Leung Amy , Chen Zhuo , Senapati Parijat , Stapleton Kenneth , Oh Hyungjiung , Kato Mitsuo , Wang Mei , Lanting Linda , Guo Qianyun , Zhang Xinyue , Zhang Bin , Zhang Haitong , Zhao Qinghao , Wang Wei , Wu Yongjian , Natarajan Rama
Published: June 2021

Angiotensin II (Ang II)‐mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in the pathogenesis of hypertension. Long non‐coding RNAs (lncRNAs) have recently elicited much interest, but their roles in Ang II actions and hypertension are not well understood. We aim to investigate the regulation and functions of a novel lncRNA “Growth factor‐ and pro‐Inflammatory cytokine‐induced Vascular cell‐Expressed lncRNA (Giver)”, in Ang II‐mediated VSMC dysfunction. RNA‐seq and RT‐qPCR analyses revealed that Ang II stimulation of rat VSMC increased expression of Giver, and Nr4a3, an adjacent gene which encodes a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with Ang II. Giver and Nr4a3 were also significantly induced by key mitogenic and pro‐inflammatory stimuli. RNA‐FISH and nuclear fractionations showed that Giver is localized predominantly in the nucleus. Regulation of Giver promoter in response to Ang II occurs through the binding of Nr4a3 to a consensus NBRE binding site. In vitro RNA pull down assays using biotinylated Giver combined with mass spectrometry in VSMC showed that Giver interacts with nuclear and chromatin remodeling related factors like ruvB‐like1, WD repeat‐containing protein 82, histone‐binding protein RBBP7, non‐POU domain‐containing octamer‐binding protein and other RNA binding proteins involved in splicing and RNA processing. Furthermore, microarray profiling combined with RT‐qPCR validation revealed that Giver knockdown attenuates the expression of genes involved in oxidative stress (Nox1) and inflammatation (Il6, Ccl2, and Tnf), but increases Nr4a3, in VSMC. Conversely, Giver overexpression enhanced the expression of these genes. Accordingly, Giver knockdown also inhibited Ang II‐induced oxidative stress and proliferation. Moreover, Giver overexpression in VSMC enhanced enrichment of Pol lI but decreased repressive histone modification H3K27me3 at oxidative stress and inflammatory gene promoters. Human orthologous genes GIVER and NR4A3 were also induced by Ang II in human VSMC and endothelial cells. Interestingly, GIVER and NR4A3 were also increased in arteries from hypertensive patients, but attenuated in hypertensive patients treated with Angiotensin Converting Enzyme Inhibitors or Angiotensin Receptor Blockers. Thus, LncRNA Giver and its regulator Nr4a3 are important players in Ang II‐mediated VSMC dysfunction, and can serve as novel targets for anti‐hypertensive therapy.

Support or Funding Information

NIH, NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic equilibrium of eEF‐2K and CaM as a regulatory logic circuit: investigations in MCF10A cells
Authors: Dalby Kevin N. , Giles David H. , Crittenden Chris M. , Kaoud Tamer S. , Ghose Ranajeet , Brodbelt Jennifer
Published: June 2021

Eukaryotic elongation factor 2 kinase (eEF‐2K) is a Ca2+/Calmodulin (CaM)‐dependent kinase that phosphorylates eEF‐2 and inactivates it, thereby blocking translation. Studies have correlated eEF‐2K expression with poorer outcomes in cancer, and it was recently shown to contribute to the Warburg effect. eEF‐2K is activated by the PKA and AMPK pathways, and inhibited by the ERK, mTORC1, and p38∂ pathways, among others. How eEF‐2K integrates these signals into a coherent output is not understood. We recently showed that phosphorylation of eEF‐2K on Ser500 by PKA, like Ca2+, drives the association of eEF‐2K and CaM to activate the kinase. We hypothesized that other regulatory signals converge on the equilibrium of eEF‐2K and CaM, either driving it toward the bound (fully active) or unbound (fully inactive) state. Here, we establish that at Ca2+ levels basal to MCF10A cells, removal of either ERK or mTORC1 activity is sufficient to fully activate eEF‐2K; increasing intracellular Ca2+ with ionomycin is sufficient to overcome these inhibitory signals. Once mTORC1 activity is already removed, additionally increasing intracellular Ca2+ produces no increase in eEF‐2K activity. Thus, inhibitory signals maintain eEF‐2K in the unbound (inactive) state, but sufficiently high Ca2+ can overcome this repression. Blocking glycolysis with 2‐deoxyglucose (2‐DG), which induces full eEF‐2K activation, results in the shutdown of ERK and mTORC1, increases intracellular Ca2+, and activates AMPK. Although AMPK phosphorylates eEF‐2K directly at Ser398, this has no effect on its association with CaM and is not required for activation by 2‐DG in cells. Similarly, directly activating AMPK (~7‐fold) with A769662 only weakly activates eEF‐2K (<2‐fold), and has no effect on ERK, mTORC1, or Ca2+. We show that, to a first approximation, the activation status of eEF‐2K is described by a simple Boolean “logic circuit”, where inhibitory inputs act to maintain the circuit in the OFF (unbound) state, and activating ones switch the circuit to the ON (CaM‐bound) state. AMPK appears to act on the circuit's inputs rather than forming a critical component itself. Further, we discuss how this “logic circuit” can be generalized to other cell types and ultimately reveal the mechanisms underpinning the regulation of eEF‐2K in cancer.

Support or Funding Information

R01 GM123252, F‐1390 Welch Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assay Development to Probe Enzymatic Heterocyclization by Fluorescence
Authors: MacRae Mark , Dieu Viet , Patterson MacKenzie , Henriquez Bryan , Soule Jessica , Xia Yuan , Dowling Daniel P.
Published: June 2021

Many therapeutically useful natural products contain 5‐membered heterocycles derived from either serine, cysteine or threonine, and important examples include the anticancer epothilones and bleomycin. In nonribosomal peptide synthetase systems, formation of these rings is catalyzed by a specific functional domain, the cyclization (Cy) domain, which catalyzes both a bond‐forming condensation reaction and a subsequent cyclodehydration reaction. To probe the reaction mechanism of this system, we are exploring the Cy domain from the protein EpoB from Sorangium cellulosum as a model system. The EpoB Cy structure has been previously characterized and determined to contain two active site channels acting as binding sites for substrate T domains. Here, we report the development of a fluorescence plate‐reader assay to monitor the cyclization reaction. Using this assay, we are exploring the functional role of residues within the Cy domain active site.

Support or Funding Information

National Institute of General Medical Sciences of the National Institutes of Health Award Number 1R15GM123425‐01 to D.P.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for direct interaction between RNA polymerase and the small ribosomal subunit.
Authors: Blaha Gregor , Diggs Stephen , Conn Adam B. , Williams Preston , Wang Yinsheng
Published: June 2021

In E. coli, RNA polymerase can directly bind ribosomes. This direct interaction may enable the coordination of the RNA polymerase and ribosome activities during transcription‐translation coupling. The coupling of both processes is exploited for gene regulation to enable immediate response to environmental changes.

To establish transcription‐translation coupling, small ribosomal subunits have to be recruited to the nascent RNAs to initiate translation. Here, we report the recruitment of the small ribosomal subunit by RNA polymerase. Similar to the binding of ribosomes to RNA polymerase, the small ribosomal subunit binding to RNA polymerase is modulated by the conformation and functional state of both macromolecules. The RNA polymerase binding site on the small ribosomal subunit was identified by chemical crosslinking and is centered on the mRNA exit site of the small ribosomal subunit.

Support or Funding Information

University of California, Riverside (start‐up funding)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.