bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanistic Study of ERCC6/CSB Enzymatic Regulation, and Its Roles in Transcription Elongation, and Chromatin Remodeling
Authors: Wang Dong 
Published: November 2023

Cockyane Syndrome Group B protein (CSB/ERCC6), a highly conserved protein from yeast to human, belongs a member of a subfamily of SWI2/SNF2‐related chromatin remodelers. CSB is proposed to be among the first proteins to be recruited to the DNA lesion‐arrested RNA polymerase II (Pol II) and initiates a special DNA repair pathway, termed transcription‐coupled repair. In addition, CSB also plays crucial roles in regulating transcription elongation and maintenance of chromatin integrity. Human CSB mutations are associated with Cockayne syndrome, a severe autosomal‐recessive disorder clinically characterized by photosensitivity, physical and mental retardation, and premature aging. However, several important mechanistic questions remain elusive: 1) how the CSB enzymatic activities are regulated for its diverse biological functions; 2) how CSB interacts with Pol II transcriptional machinery; 3) What is the molecular mechanism of CSB chromatin remodeling.Recently, we identified a novel conserved protein motif, termed the “leucine latch”, which plays a key role in autoregulation of CSB enzymatic activities. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 in spite of the otherwise highly divergent nature of the N‐ and C‐terminal regions. This work provides important novel mechanistic insights into the autoregulation of CSB/ERCC6 proteins. We will also present our unpublished results toward the mechanistic understanding of roles of CSB in transcription elongation, and chromatin remodeling.Support or Funding InformationNIH GM102362
Chromatin Remodeler EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation
Authors: Pradhan Suman Kalyan , Su Trent , Yen Linda , Jacquet Karine , Cote Jacques , Kurdistani Siavash , Carey Michael 
Published: November 2023

Increasing evidence indicates that gene activation in metazoans is accompanied by deposition of the histone variants H2AZ and H3.3 into promoters and enhancers. The abundance of double variant nucleosomes correlates well with the levels of gene expression. However, knowledge is limited for how variant chromatin is assembled in regulatory regions and, more importantly, whether it plays a direct role in gene activation. To unravel the mechanism, we have recreated activator‐ and HAT‐stimulated transcription from H3.3/H2AZ variant chromatin in HeLa nuclear extracts. We further employed Immobilized template assays to identify variant chromatin specific chromatin proteins. Analysis of preinitiation complex (PIC) revealed that the chromatin remodeling factor EP400 was specifically enriched on variant versus canonical chromatin. In a cell based assay, EP400 was recruited to an integrated inducible promoter in U2OS cells in response to gene activation. Moreover, siRNA knockdown of EP400 led to diminished transcription and reduced assembly of H3.3‐containing chromatin, while having little or no effect on binding of the Mediator co‐activator. Remarkably, this observation was paralleled genome‐wide at promoters and enhancers, where knockdown of EP400 significantly affected H3.3 deposition, but not Mediator recruitment. We investigated the mechanism by in vitro histone exchange assay. EP400 is known to exchange H2AZ for H2A but in biochemical histone exchange assays, it also inserted H3.3 for H3.1 in a nucleosome dependent manner. Interestingly, a side‐by‐side comparison of histone exchange assay indicates that reverse exchange, i.e. exchange of canonical histone into variant chromatin is much less efficient. However, H3.3 exchange into variant chromatin is highly efficient. We reason that this dynamic exchange of H3.3 with H3.3 may play a role in enhaced transcription. What confers this specificity remains an open question. To this end we are currently exploring the contribution of Brd8, a bromodomain containing protein that resides in the same complex and happens to interact directly with EP400. Indeed, we have preliminary evidence from modified histone peptide array experiments that suggest that Brd8‐EP400 duo is significantly enriched in H3K18ac and H3K27ac histone marks. This raises further likelihood of collaboration between EP300 and EP400 in the context of gene activation. Our data support a model in which EP400 links variant chromatin assembly directly to gene activation but downstream of PIC assembly in a pathway parallel to pre‐initiation complex assembly but necessary for transcription on chromatin.Support or Funding InformationL.Y. is supported by the Ruth L. Kirschstein National Research Service Award GM007185
Circadian lnc to EDN1‐AS: A Novel, Long, Non‐Coding RNA Antisense to the EDN1 Gene
Authors: Solocinski Kristen , Barilovits Sarah J. , Welch Amanda K. , Wingo Charles S. , Cain Brian D. , Gumz Michelle L. 
Published: November 2023

Endothelin‐1 (ET‐1) is a peptide signaling molecule that acts as a potent vasoconstrictor in the vasculature whereas in the kidney it has natriuretic (sodium‐excreting) effects. ET‐1 is a critical regulator of blood pressure and dysregulation of ET‐1 signaling can result in several pathologies including hypertension and chronic kidney disease. It is widely accepted that the main mechanism for regulation of ET‐1 expression is through transcriptional control of the gene that encodes ET‐1, Edn1. However, we have discovered a long, non‐coding RNA (lncRNA) antisense to the Edn1 gene, called EDN1‐AS, which represents a putative mechanism for regulation of ET‐1 expression. Strand‐specific RT‐PCR has demonstrated that EDN1‐AS is present in multiple human cell lines as well as authentic human kidney RNA. We have also detected EDN1‐AS in mouse kidney and aorta. Preliminary data indicate possible sex and localization differences in EDN1‐AS expression. Expression also seems to be regulated by the circadian clock as EDN1‐AS expression oscillates in human kidney proximal tubule (HK2) cells synchronized with dexamathasone. EDN1‐AS expression is conserved in both humans and mice and appears to be regulated in multiple ways. Therefore, we hypothesize that EDN1‐AS represents a novel regulatory mechanism of ET‐1 expression and may present a novel therapeutic target for diseases caused by aberrant ET‐1 expression.Support or Funding InformationThis work was supported by NIH DK085193 and DK098460, the ASN Foundation for Kidney Research to MLG, 2T32HL083810 to KS, 2T32HL083810 to AKW, and NIH DK082680 to BDC and CSW.
Editing of Kv1.1 RNA Transcripts is Altered by Human Mutations Respons‐ible for Episodic Ataxia Type‐1
Authors: Kiddie Elizabeth Ferrick , Patterson Kathleen M. , Reenan Robert A. , Rosenthal Joshua J. C. , Emeson Ronald B. 
Published: November 2023

Transcripts encoding the Kv1.1‐subtype of voltage‐gated potassium channel un‐der‐go an adenosine‐to‐inosine (A‐to‐I) RNA editing event resulting in the con‐ver‐sion of a genomically‐encoded isoleucine (AUU) to a valine codon (IUU) at amino acid position 400 (I400V). Previous studies have shown that this single amino acid change alters the recovery rate following channel inactivation and has been pro‐posed to mod‐u‐late the kinetics of neuronal repolarization due to its location within the predicted ion pore of the channel. Numerous nonsense and mis‐sense muta‐tions in the Kv1.1 gene (kcna1) have been associated with the hu‐man po‐tas‐sium channelopathy, Episodic Ataxia type‐1 (EA1), which is char‐ac‐ter‐ized by epi‐sodes of stress‐induced ataxia and myokymia, and can be associated with epi‐leptic seizures. Three human EA1 mu‐ta‐tions (V404I, I407M, and V408A) are within close prox‐imity to the I400V editing site, and are contained within the RNA duplex structure required for A‐to‐I con‐ver‐sion. We have employed both in vitro and in vivo systems to determine the impact that these EA1 mutations might have on Kv1.1 RNA editing, as well as determined the electro‐phys‐iological changes in chan‐nel function derived from tran‐scripts containing EA1 mutations with either an edited or non‐edited status. In vitro tran‐scribed RNA substrates containing EA1 mutations were incubated with nuclear extracts isolated from cells overexpres‐sing the ADAR2 editing enzyme. Each of the EA1 mu‐ta‐tions dif‐fer‐en‐tial‐ly de‐creased A‐to‐I conversion of Kv1.1 RNAs in relation to their over‐all prox‐im‐ity to the editing site. Furthermore, a mouse model of EA1, encoding the human V408A mutation at the endogenous kcna1 locus, also re‐vealed a significant decrease in Kv1.1 editing in all tissues examined. Analysis of EA1 mutant chan‐nels using a Xenopus oocyte expression system, in a non‐edited (I400) or edited (V400) background, revealed significant changes in the elec‐‐tro‐physiological char‐ac‐teristics of the channel. These observations suggest that altered Kv1.1 function in EA1 could result from both the missense mutation and alterations in editing ef‐fici‐ency. These results also are con‐sis‐tent with a gen‐et‐ically‐modified mouse mod‐el of Kv1.1 editing, where mice sole‐ly expressing the non‐edited Kv1.1 iso‐form exhibit stress‐in‐duced ataxia and an increase in induced‐sei‐zure suscepti‐bil‐i‐ty.Support or Funding InformationJoel G. Hardman Chair in Pharmacology (RBE)Multidisciplinary Training in Molecular Endocrinology (METP) NIH/NIDDK – T32 DK007563 (EFK)Ruth L. Kirschstein National Research Service Award NINDS F31 NS087911 (EFK)
RNA Editing‐Mediated Modulation of Calcium‐Dependent Activator Protein for Secretion 1 (CAPS1) Function
Authors: Emeson Ronald B , Ulbricht Randi J , Lazarenko Roman M , DelBove Claire E , Zhang Qi 
Published: November 2023

CAPS1 is a priming factor, involved in the regulated release of hormones, peptides and neurotransmitters from large dense‐core secretory granules and synaptic vesicles. Tran‐scripts encoding CAPS1 are modified by an adenosine‐to‐inosine (A‐to‐I) RNA editing event in which a genomically‐encoded glutamate (GAG) is converted to a glycine (GIG) codon. This non‐synonymous amino acid change is located within the essential carboxyl‐terminal domain of the protein which mediates the CAPS1 interaction with peptide and hormone‐containing vesicles, suggesting that RNA editing modulates the contribution of CAPS1 to evoke vesicular release. Editing of CAPS1 was found to be conserved in vertebrate species from humans to zebrafish, indicating its importance in regulated secretion, an essential cellular function in the nervous and endocrine systems. Quantitative analysis of editing for CAPS1 transcripts in dissected mouse brain regions and peripheral endocrine organs revealed that editing was highest in the cortex of the brain (~24%) and lowest in the testes (~2%). Furthermore, the sequences necessary for editing were found to be contained within a predicted duplex region of the pre‐mRNA formed by two halves of a 32 base‐pair inverted repeat surrounding the editing site (exon 27) and within intron 27. Electrophysiological analyses of primary neuronal cultures from postnatal rat hippocampus revealed that neuronal synapses were enhanced by overexpression of the non‐edited CAPS1 isoform. Surprisingly, FM 1–43 dye loading and unloading studies suggested that synaptic vesicle release and retrieval were less complete with the non‐edited CAPS1 isoform. Tracking the fate of synaptic vesicle protein recycling during exocytosis and endocytosis, using a red fluorescence pH‐sensor (pHTomato) fused to the synpatophysin luminal domain, revealed that the non‐edited CAPS1 isoform promoted fast, but incomplete, fusion (i.e. kiss‐and‐run) that is often exerted by docking vesicles. These data suggests that CAPS1 editing may serve as a molecular mechanism to fine tune synaptic connectivity through less appreciated vesicle docking and recycling kinetics at pre‐synaptic terminals. These studies provide insights into the functional importance of CAPS1 editing in the regulation of neuronal network connectivity, as well as suggest a broader role for CAPS1 RNA editing in vertebrate physiology.Support or Funding InformationJoel G. Hardman Chair in Pharmacology (RBE) and NIH OD008761 (QZ)
Phosphorylation‐independent recruitment of 14‐3‐3 to tristetraprolin partially impairs deadenylation by blocking Ccr4, but not Pan2, association
Authors: Nunez Castrejon Ana M. , Tun Maria , Nava Raul , Kraemer Laura , Clement Sandra L. 
Published: November 2023

The regulation of mRNA stability by RNA binding proteins plays a key role in the modulation of gene expression. The RNA‐binding protein tristetraprolin (TTP) binds to AU‐rich elements (AREs) in the 3′ untranslated regions (UTRs) of transiently expressed mRNAs encoding cytokines, growth factors, and proto‐oncogenes and stimulates their decay by recruiting a number of mRNA decay enzymes including the Ccr4 and Pan2 deadenylase complexes. Phosphorylation of serines 52 and 178 of TTP by the p38‐activated MAP kinase MK2 impairs TTP's ability to stimulate mRNA decay by preventing the recruitment of these deadenylases. Phosphorylation of TTP also creates a binding site for the protein 14‐3‐3, although it is unclear whether 14‐3‐3 association is required to impair TTP function. In order to determine whether 14‐3‐3 association is sufficient to prevent deadenylase recruitment in the absence of phosphorylation, we replaced serines 52 and 178 in TTP via site‐directed mutagenesis with either a peptide sequence known to bind 14‐3‐3 independently of phosphorylation or with an altered version of this peptide sequence that does not. The ability of Myc‐tagged wild‐type and mutant forms of TTP to bind FLAG‐tagged Ccr4, Caf1, or Pan2 deadenylases was tested in anti‐FLAG co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. Mutant forms of TTP that bound 14‐3‐3 independently of phosphorylation were unable to interact with the either the Ccr4 or Caf1 proteins but retained their ability to associate with Pan2 whereas forms of TTP that failed to bind 14‐3‐3 bound all three proteins. These results suggest that 14‐3‐3 association with TTP independently of phosphorylation does not fully recapitulate the effect of phosphorylation.Support or Funding InformationThis project was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant to SLC. ANC received a stipend from the Cal Poly College of Science and Mathematics 2015 Summer Research Program. MT received a stipend and research funds from the Allan Hancock College/Cal Poly SLO Bridges to the Baccalaureate program funded by the National Institutes of Health.
Effects of Aging and Nitrogen Deprivation on Nonfunctional rRNA Decay in Saccharomyces cerevisiae
Authors: Zekan David , LaRiviere Frederick J 
Published: November 2023

Nonfunctional Ribosomal RNA Decay (NRD) is a quality control pathway in Saccharomyces cerevisiae capable of degrading functionally defective mature rRNAs. Two types of NRD are known: 18S NRD which removes rRNAs with mutations in the small subunit decoding site and 25S NRD which removes rRNAs with mutations in the large subunit peptidyl transferase center. 18S NRD is translation dependent and occurs after ribosomal subunit joining on the mRNA. 25S NRD is mechanistically distinct; 25S rRNA degradation is translation independent and occurs prior to ribosomal subunit joining. We are interested in understanding this pathway because rRNAs are usually very abundant, generally comprising greater than 80% of the total cellular RNA, and very stable, with half‐lives on the order of several days. Therefore, natural NRD substrates in the form of damaged rRNAs may increase as cells age. Moreover, defective rRNAs may accumulate in NRD‐deficient cells. General ribosome turnover also increases under certain cellular stress conditions. For example, nitrogen deprivation results in degradation of both small and large ribosomal subunits by ribophagy. However, the effect of nitrogen starvation on NRD is unknown. Therefore, we are interested in analyzing the effects of both chronological aging and nitrogen deprivation on 18S and 25S NRD. To do this, we are measuring growth phenotypes and rRNA expression levels in yeast grown in normal or nutrient‐deprived media for various lengths of time. These experiments use either wild‐type or NRD‐deficient yeast strains transformed with rRNA expression plasmids that encode functional and/or nonfunctional versions of the 18S and 25S rRNAs. The plasmid‐derived rRNAs also contain short, unique sequence tags that allow for specific detection of these rRNAs in the presence of total cellular rRNA. Data collection and analysis are currently ongoing. We hope that these studies might provide insight into the accumulation of defective rRNAs over time in chronologically aged cells and the effects of nitrogen deprivation on this novel rRNA decay pathway.Support or Funding InformationThis work is supported by the Dr. G. Ashley Allen Student Research Grant at Washington and Lee University.
Characterization of the ubiquitin binding properties of the deadenylase Pan2
Authors: Swinney Zachary , Teruya Jonathan , Vengoechea Nicole , Clement Sandra L. 
Published: November 2023

Messenger RNA (mRNA) stability plays an important role in regulating gene expression in eukaryotic cells. While a number of endo‐ and exo‐nucleolytic enzymes act on mRNA, removal of the poly(A) tail, or deadenylation, is often the first and rate‐limiting step in mRNA decay. This process is initiated by the Poly(A) Nuclease (Pan2) complex and completed by the Ccr4/Caf1/Not complex(1). In addition to its poly(A) specific exonuclease domain, the Pan2 protein also contains a conserved domain belonging to a class of deubiquitinating enzymes (DUBs). Amino acid sequence alignments of Pan2 with other DUBs show that two of the three essential amino acids required for catalysis are not conserved, making it unlikely that Pan2 retains the ability to remove ubiquitin. However, the strong conservation of residues known to play a role in ubiquitin binding suggests that Pan2 might have retained its ability to bind ubiquitin. In order to test whether Pan2 binds ubiquitin, we performed a ubiquitin binding assay by incubating ubiquitin‐agarose resin with FLAG‐tagged Pan2 expressed in transiently transfected Human Embryonic Kidney (HEK) 293T cells and observed that Pan2 binds to ubiquitin‐agarose, but not to agarose lacking ubiquitin. The ability of Pan2 to bind ubiquitin agarose was abolished by pre‐incubation of cell lysates with excess amounts of free ubiquitin. In order to identify ubiquitinated Pan2 binding partners, anti‐FLAG co‐immunoprecipitations are currently being performed in lysates from cells co‐expressing FLAG‐tagged Pan2 and Myc‐ubiquitin‐tagged proteins. The observation that Pan2 binds ubiquitin raises the intriguing possibility that mRNA deadenylation by Pan2 may be regulated by ubiquitin‐dependent protein interactions.Support or Funding InformationThis work was supported in part by a grant to S.L.C. from the Cal Poly Extramural Funding Initiative Program.
Exploring GTP Control of Bacterial Respiratory System Specific Chaperone Interaction with its Substrate Protein's Twin‐Arginine Signal Peptide
Authors: Cherak Stephana Julia , Turner Raymond J 
Published: November 2023

Many bacterial respiratory redox enzymes depend on the twin‐arginine translocase (Tat) system for translocation and/or membrane insertion. Tat substrates contain an N‐terminal twin‐arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone ‐ redox enzyme maturation protein (REMP) – to help mediate final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin‐arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase (DmsABC). DmsD adopts monomer, dimer, and unique folding forms that appear under pH control. All alternate DmsD forms retain the ability to bind DmsAL. As well, it is now suggested that DmsD cooperatively binds guanosine‐5′‐triphosphate (GTP), which elicits a dissociative effect on the DmsD to DmsAL interaction. Thus, the primary aim of this study was to analyze DmsD thermal stability when subjected to both GTP association and variance in pH. We have utilized two in vitro protein‐protein interaction techniques of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF). DSF experiments, yielding melting temperatures as a determinant of the associated thermal stability, showed an increase in DmsD stabilization upon DmsAL binding. Between pH values of 5.0 and 6.0, DmsD demonstrated the least amount of DmsAL release. This pH range also corresponds to a transition in DmsD folding forms. When DmsD was first subjected to GTP binding, subsequent variance in pH decreased melting cooperativity by the appearance of a pretransition peak, but did not affect melting temperature. However, if alteration in pH occurred prior to GTP binding, the pretransition peak was attenuated, and an incremental increase in melting temperature was observed. These findings illustrate that a greater affinity for DmsAL promotes an increase in DmsD thermal stability. As well, pH shift results in formation of a more uniform DmsD population, with subsequent GTP binding permitting an increase in thermal stability. These results support the idea that H+ and GTP play roles as molecular regulators in the biochemical pathway of DmsAB protein maturation – Tat system targeting mechanism.Support or Funding InformationResearch was supported by a Canadian Institutes of Health Research operating grant (MOP‐49422) to RJT.
Prion ‐ like protein polymerisation underlies signal transduction in innate immunity: the emergence of a universal mechanism ?
Authors: O'Carroll Ailis , Ve Thomas , Lau Derrick , Giles Nichole , Moustaqil Mehdi , Bhumkar Akshay , Hunter Dominic , Kobe Bostjan , Boecking Till , Sierecki Emma , Gambin Yann 
Published: November 2023

The innate immune system uses a multitude of receptors such as Toll‐like Receptors and Nod‐like Receptors to sense pathogens and danger signals. Upon their activation, a signalling cascade is initiated culminating in the transcription and translation of cytokines and other proteins vital for host protection. Recent studies have discovered that an array of these intracellular signalling proteins assemble into oligomeric signalling platforms, consequently amplifying the transmission of the signal from the stimulated receptor to the activation of relevant transcription machinery.Unexpectedly, it was also discovered that some of these proteins present in innate immune signalling such as the inflammasome adaptor protein ASC and the mitochondrial membrane‐associated protein MAVS, can form fibrils. These fibrils can elongate in a prion‐like manner to create super‐sized signalling platforms.In this study, we screened over 100 innate immune intracellular signalling proteins using single molecule fluorescence. We defined the oligomerisation and aggregation propensity of these proteins and track the specific signature of polymerisation. We identified multiple candidates forming super‐sized protein aggregates; through protein seeding experiments, we were able to conclude that ~20 of these proteins could self‐propagate aggregation as found in prion‐like mechanisms.These innate immune cytosolic proteins are not isolated to one signalling pathway but transverse most, if not all, signalling pathways of innate immune defence, inflammation and cell death. Intriguingly, some of these proteins could act to regulate and even inhibit the signalling cascade through their prion‐like protein propagation.Together these findings reveal a universal mechanism where the creation of polymer‐like platforms increases the number of binding sites and dramatically amplifies the signal. Our observations also suggest a complex interplay among the major signalling cascades of the innate immune system, in which prion‐like polymerization underlies the backbone of intracellular communication.