bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationFunded by National Basic Research Programs of China, No.2014CB744404This 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 InformationNIH DP1HD087990This 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 InformationFunds from the Department of Natural Sciences and the Office of Research and Sponsored Programs at the University of Michigan DearbornThis 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 InformationMSU Denver Applied Learning Center Undergraduate Research Grant, Colorado‐Wyoming Alliance for Minority Participation Undergraduate Research Grant, MSU Denver Dean's GrantThis 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 InformationNIH, NHLBIThis 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 InformationR01 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 InformationNational 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 InformationUniversity 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.
Investigating Carrier Domain Positioning During Catalytic Turnover in Pyruvate Carboxylase
Authors: Hakala Joshua , St. Maurice Martin 
Published: June 2021

A central debate in protein biochemistry focuses on the mechanism by which ligand binding correlates with protein conformational change. Two primary hypotheses describe this process: the induced‐fit hypothesis states that ligand binding induces a conformational change in the protein, while the conformational ensembles hypothesis states that the protein pre‐exists in multiple conformational states, with ligand binding shifting the equilibrium towards a preferred conformation. Pyruvate carboxylase (PC) is a multi‐domain, swinging‐arm enzyme that coordinates reactions between two remote active sites and serves as a structurally and biophysically accessible paradigm to study the relationship between ligand binding and protein conformational change. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. The biotin cofactor on the biotin carboxyl carrier protein (BCCP) domain is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then translocates to the carboxyltransferase (CT) domain on an opposing subunit, where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate.In order to better understand the mechanism of carrier domain translocation, the equilibrium positioning of the BCCP domain was directly observed using site‐specific cross‐linking to trap the BCCP domain in the putative exo‐binding site on the opposing subunit. This approach permits a detailed analysis of BCCP domain positioning in response to various substrates and allosteric effectors. Samples were first analyzed using SDS‐PAGE and densitometry to measure BCCP cross‐linked dimers as reporters for equilibrium positioning changes. Given that BCCP cross‐linking inactivates the enzyme, rates of inactivation were measured to assess changes in BCCP domain positioning in the presence of substrates and effectors. Finally, intrinsic tryptophan fluorescence changes were measured on fast timescales to more accurately determine the effect of ligands on the rates of BCCP domain repositioning. These studies reveal that substrates and effectors shift the BCCP domain positioning from one domain to another and offer the first evidence that the carrier domain exists in a dynamic equilibrium between active sites.Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.