bluuebunny/crossref_metadata_2025 huggingface dataset:
Complementary roles of Pseudomonas aeruginosa condensins in global chromosome organization
Authors: Bhowmik Bijit Kumar , Rybenkov Valentin V
Published: November 2023

Condensins play a crucial role in global chromosome maintenance and organization in all forms of life. In bacteria, condensins contribute to proper resolution and segregation of sister chromatids following replication. Atypically, Pseudomonas aeruginosa strain PAO1 expresses two condensins, the canonical SMC‐ScpAB complex and the unconventional MksBEF. These two proteins belong to two different super‐families of condensins. Here we investigated the roles of SMC‐ScpAB and MksBEF in chromosome segregation of PAO1.

Pseudomonas aeruginosa condensin mutants, ΔmksB and Δsmc were constructed using homologous recombination. To visualize different loci on the chromosome, arrays of tetO operators were inserted into pre‐determined positions of P. aeruginosa chromosome and used as a binding target for TetR‐CFP chimera. TetR‐CFP was expressed from a plasmid under an IPTG inducible lacUV5 promoter. Individual chromosomal loci were then observed using fluorescence microscopy while cellular morphology was characterized using phase‐contrast microscopy

PAO1 chromosome segregation initiated from a unique origin (oriC), progressed bidirectionally and terminated at the site‐specific recombination site dif, responsible for the resolution of daughter chromosomes. Notably, the oriC and dif sites are asymmetrically positioned on the PAO1 chromosome. As a result, the left arm is about 1.6 times longer than the right arm. Thus, segregation along the arms proceeds with unequal rates.

In newly born cells, the oriC was located close to mid‐cell, whereas dif site was located close to the new pole. Following replication, oriC split into two foci, relocated to the quarter positions and remained there for the rest of cell cycle. In contrast, dif site was pulled from the pole, towards the mid‐cell and split at the very end of cell cycle. We found two macrodomains on the left arm of the chromosome. Deletion of mksB did not change the longitudinal organization of PAO1 chromosome. At the same time, the sequence of chromosome segregation remained unchanged in ΔmksB cells. However, the timing of segregation of chromosomal loci were significantly altered. Interestingly, one of the macrodomains was dispersed in ΔmksB cells. Deletion of smc had a more dramatic effect. It markedly altered segregation of oriC while most of the chromosome was disorganized

Our findings indicate that both SMC and MksB contribute to the global chromosome organization in P aeruginosa. SMC and MksB has complementary but non‐overlapping roles in chromosome segregation. While SMC is required for timely segregation of oriC region, MksB ensures compactness of the chromosome.

Support or Funding Information

This work was supported by the award OCAST HR 14–042.
Methylation status of the gene encoding Akt2 in cinnamon extract treated MCF‐7 cells
Authors: Hill Matthew , Bulko Rachel , Aulthouse Amy , Kinder David , Stockert Amy
Published: November 2023

The role of epigenetics in cancer development and progression has exponentially grown in recent years. Polyphenolic compounds from green tea and other sources have been demonstrated to alter methylation status in a variety of genes, including those linked to cancer. Our studies suggest similar results may be possible from treatment of cells with cinnamon extract. We selected the MCF‐7 breast cancer cell line to explore the effects of cinnamon treatment on DNA methylation patterns. Following confirmation of total methylation changes we selected genes of interest in cancer development to complete methylation specific PCR aimed at determining the effects of gene specific methylation changes in cinnamon treated cells. Akt2 signaling can play a major role in different signaling pathways to prevent cellular apoptosis and lead to prolonged cell life. Research also demonstrates variable localization of the Akt2 protein, with confirmed identification in both the cytoplasm and the nucleus. Preliminary results in the lab demonstrate the potential for aqueous cinnamon extract to methylation the Akt2 gene in MCF‐7 cells, potentially decreasing expression of the protein. Here we will present our results demonstrating the methylation change on Akt2 upon cinnamon extract treatment as well as further characterize effects of cinnamon on Akt2 expression and localization. Collection of treated and untreated cells under various treatment regimes will allow further methylation analysis, RNA extraction followed by quantitative PCR to evaluation expression of Akt2. Additionally we will perform Western blot analysis on cytoplasmic and nuclear proteins to determine both protein expression and localization.

Support or Funding Information

Departments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University
MCF‐7 Morphological changes upon treatment with aqueous cinnamon extract
Authors: Bulko Rachel , Aljahani Rawan , Aulthouse Amy , Kinder David , Stockert Amy
Published: November 2023

Epigenetics has emerged as a key component in both the prevention and the development of various cancers. Polyphenolic compounds from a variety of sources have been explored as potential epigenetic modifiers for both methyaltion and acetylation. Furthermore, studies exploring extracts versus individual components have demonstrated up to 50% more activity when treating cells with the full extract rather than select components. Our studies suggest a similar synergistic effect when treating MCF‐7 breast cancer cells with aqueous cinnamon extract. These cells exhibit a proliferative response when exposed to heightened endogenous estrogen and progesterone. Upon administration of certain natural substances, however, proliferation can be lowered significantly. Simulating potential cancer treatments in vitro can be accomplished with this cell line, since it has characteristics of metastatic breast cancer. Visible changes in cell morphology, including shrinking and becoming rotund, can be observed in the MCF‐7 cells treated with cinnamon extract. Similar results have been obtained from trials involving turmeric and curcumin, which researchers believe may be due to the qualities of the polyphenol levels that exist in these organic compounds. Here we present results of a morphological study involving treatment of MCF‐7 cells with cinnamon extract and or various components identified in the extract. We report changes in cell shape, with treated cells adopting a round shape rather and polygonal which is not induced by changes in monolayer attachment. We also see a significant difference in cell size upon cinnamon treatment. We explored changes in methylation patterns from extracted DNA and selected target genes of interest, FoxO1 and GSK3beta for anaylsis by methylation specific PCR.

Support or Funding Information

Departments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University.
Combinatorial Effects of PK11195 and 5‐Azacytidine as an Epigenetic Modulator For MPC1
Authors: Duran Daniel , O'Donnell Robert Watts
Published: November 2023

According to the Warburg effect, cancer cells typically favor glycolysis over oxidative phosphorylation wherein only a few select molecules of pyruvate are oxidized. The mitochondrial pyruvate carrier, MPC, contributes significantly to proper cellular respiration and its under expression correlates strongly with the observed “fractional pyruvate oxidation.” The associated MPC1 gene is under expressed or even deleted across multiple tumor cell lines, contributing to their preference of glycolysis as an energy supplying mechanism. We hope to reverse the Warburg effect by enhancing MPC1 gene expression and thereby increase the potential for tumor cells to undergo oxidative phosphorylation following glycolysis. Use of the DNA methyltransferase inhibitor 5‐azacytidine may, through its role as an epigenetic modulator, contribute to enhanced expression or even re‐expression of the MPC1 gene. An additional drug, PK11195, was chosen for its role as a peripheral benzodiazepine receptor ligand to similarly observe it potential effect on MPC1 expression. The drug itself binds with nanomolar affinity to the PBR on the outer mitochondrial membrane and alters mitochondrial respiration. Through qRT‐PCR we have demonstrated an increased expression of MPC1 in the HTB‐4 cell line upon co‐treatment with PK11195 and 5‐azacytidine over either drug used alone. This suggests the cells are navigating from glycolysis to an oxidative pathway in which MPC1 is an integral part. Experiments are planned to verify whether or not the MPC‐1 gene is similarly up regulated in other tumor cell lines; furthermore, we will investigate if the treated cells show an increase in reactive oxygen species, indicating a transition to oxidative phosphorylation.

Support or Funding Information

The Geneseo Foundation and the SUNY Geneseo Biology Department.
Deciphering the Dynamics of UHRF1‐dependent DNA Methylation
Authors: Tiedemann Rochelle L. , Jones Peter A. , Rothbart Scott B.
Published: November 2023

The epigenetic inheritance of DNA methylation patterning is a coordinated process involving UHRF1‐directed recruitment of DNMT1 to S‐phase chromatin, where the parental methylation patterns are copied on nascent DNA. As UHRF1 is now recognized as a key regulator of DNA methylation maintenance, it is considered as a promising therapeutic target for modulating the DNA methylation program in cancer and other diseases. UHRF1 itself is targeted to chromatin in a multivalent manner by the interconnected activities of three reader domains, which directs its ubiquitin ligase activity towards several lysine residues on histone H3 (a proposed docking site for DNMT1). While the mechanistic links between UHRF1 and DNA methylation maintenance are beginning to be unraveled, little is known about the dynamics of this process at epigenome‐scale resolution. To study the dynamics of UHRF1‐dependent DNA methylation, we engineered a human colorectal carcinoma cell line that expresses a doxycycline‐inducible shRNA targeting UHRF1. Importantly, this system is reversible, allowing us to model the effects of acute depletion of UHRF1 in these cells. Using this system, we are following UHRF1‐dependent DNA methylation dynamics using Illumina's Infinium Methylation EPIC Bead Array and layering this information with transcriptome, nucleosome occupancy, and histone modification dynamics. We are also querying the contributions of specific UHRF1 regulatory domains to the maintenance of epigenome integrity. Collectively, the results of this study are providing high‐resolution temporal snapshots of epigenetic programming (and reprogramming) facilitated by UHRF1 and are revealing how the epigenome responds to targeted inhibition of UHRF1 function.

Support or Funding Information

NIH NCI R00 CA181343 to SBR; NIH NCI R01 CA082422, R01 CA170550 to PAJ
Epigenetic Regulation of Alternative Developmental Trajectories
Authors: Romney Amie Lynn T. , Podrabsky Jason E.
Published: November 2023

The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead “escape” entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA‐seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA‐seq data during early development, after the maternal‐to‐zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal‐embryonic‐conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development.

Support or Funding Information

NSF IOS 1354549
Spatio‐temporal Dynamics of Sphingosine‐1‐Phosphate Receptor 1 Activity in Endothelial Cells During Lung Injury and Resolution
Authors: Akhter Md. Zahid , Mehta Dolly
Published: November 2023

Sphingosine‐1‐phosphate receptor 1 (S1PR1) signaling has been implicated in regulation of vascular permeability, lymphocyte trafficking, inflammation, and cardiac function. In endothelial cells (ECs), S1PR1 stimulates Gi‐dependent intracellular signaling cascades, which enhances barrier function. ECs regulate primary host defense mechanism. In this study, we used S1PR1‐GFP signaling reporter mice to determine dynamics of S1PR1 activity in the lung in response to endotoximia during injury and resolution. These mice, having S1pr1 knockin vector crossed with histone‐GFP reporter (H2B‐GFP) mice. Activation of S1PR1 induces β‐arrestin‐tobacco etch virus (TEV) protease binding to the activated receptor and subsequent release of tetracycline transcriptional activator (tTA) leading to translocation of tTA to nucleus to drive transcription of the histone‐GFP reporter. LPS in a time dependent manner increased GFP expressing cells in the lung. Flow cytometry of GFP gated cells revealed significantly increase in ECs (CD45−CD31+GFP+) in the lungs of S1PR1 GFP signaling mice, compared to that in control H2B‐GFP mice. Whereas, LPS increased apoptosis of non‐GFP+ cells at 16 h, S1PR1 activated ECs remain viable. Immuno‐histological detection of GFP+ nuclei in lung tissue section revealed localized activation of S1PR1 during injury and repair. Studies are underway to characterize the endothelial cells (ECs) showing S1PR1 activity versus control ECs to assess if S1PR1 activity in ECs is a potential determinant of EC‐regeneration.
Nuclear export factor 3 regulates localization of snoRNAs
Authors: Li Melissa , Lee Jiyeon , Sletten Arthur , Pyles Kelly , Schaffer Jean
Published: November 2023

Small nucleolar RNAs (snoRNAs) guide chemical modifications of ribosomal and small nuclear RNAs and canonically localize in the nucleus. Recent studies have demonstrated that some snoRNAs have broader cellular roles, such as a function for ribosomal protein L13a (Rpl13a) snoRNAs in lipotoxic and oxidative stress responses. The observation that these and other snoRNAs are present in the cytoplasm under homeostatic conditions and accumulate in the cytosol during metabolic stress suggests that localization of snoRNAs is tightly regulated. However, there is no known nucleocytoplasmic transporter for snoRNAs. Using an shRNA loss‐of‐function genetic screen in endothelial cells, we found that an shRNA targeting nuclear export factor 3 (NXF3), a member of the nuclear RNA export factor family, caused resistance to metabolic stress. Given that Rpl13a snoRNAs are critical for metabolic stress‐induced cell death, and that these snoRNAs accumulate in the cytoplasm under metabolic stress conditions, we hypothesized that NXF3 may function as a regulator of snoRNA distribution. In transient transfection assays, NXF3 knockdown increased abundance of cytosolic Rpl13a snoRNAs, whereas NXF3 overexpression led to decreased cytosolic levels, suggesting that NXF3 functions in nuclear import of snoRNAs. We found the Rpl13a snoRNAs co‐immunoprecipitated with NXF3, consistent with a role for NXF3 as a snoRNA transporter. Treatment of cells with forskolin, which causes a rapid decrease in cytosolic snoRNAs, increased both nuclear localization of NXF3 and association of NXF3 with Rpl13a snoRNAs. Treatment of cells with doxorubicin, which causes cytosolic accumulation of Rpl13a snoRNAs, decreased association of the snoRNAs with NXF3. Together, our findings suggest a model in distribution of snoRNAs between the nuclear and cytoplasmic compartments that is regulated by NXF3.
Differential Alternative Splicing in Clinical Strains of Cryptococcus neoformans
Authors: Merryman Mitch Carlton , McClelland Erin , Seipelt‐Thiemann Rebecca
Published: November 2023

Alternative splicing allows eukaryotes to produce a larger number of proteins from a limited number of genes. Alternative splicing has also been associated with virulence in pathogenic eukaryotes, such as fungi like Cryptococcus neoformans. It is expected that clinical strains of C. neoformans will exhibit differing patterns of alternative splicing in virulence factor genes than less‐pathogenic laboratory strains of C. neoformans. If new isoforms of virulence factor genes could be identified in pathogenic strains, they could potentially be used as targets for new therapeutics for cryptococcosis. To more fully understand the differential patterns of alternative splicing in clinical strains and identify these potential therapeutic targets, mRNA from seven different clinical isolates and one laboratory strain, H99S, was isolated and analyzed using RNA sequencing. Initial bioinformatics analyses of the RNA sequencing data was undertaken using TopHat, Cufflinks, and Cuffdiff tools. These data will be compared to other models and tool sets including replicate multivariate analysis of transcript splicing (rMATS), Splicing Compass, Exon Quantitation Pipeline (EQP).

Support or Funding Information

Biology Department, Middle Tennessee State University

URECA
Export of discarded, splicing intermediates provides insight into mRNA export
Authors: Zeng Yi , Staley Jonathan
Published: November 2023

The precise removal of introns is essential for cell survival. To promote fidelity in nuclear pre‐‐mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates that have engaged the spliceosome. Although these discarded splicing substrates can be degraded in the nucleus, discarded splicing species, the 5′ exon and lariat intermediate, can be exported to the cytoplasm, as the discarded species can be translated and degraded by cytoplasmic nucleases. However, it is unknown how these suboptimal intermediates are exported.

Here, we used single molecule RNA‐FISH in budding yeast to provide direct evidence that cells export suboptimal splicing intermediates, both the 5′ exon and lariat intermediate. We observed that the export of suboptimal splicing intermediates requires the nuclear basket and the general mRNA export receptor Mex67. Further, we found that two essential Mex67 adaptors, Yra1 and Nab2, are required for exporting suboptimal lariat intermediates, demonstrating that both mRNAs and lariat intermediates undergo a similar process to assemble export machinery. Surprisingly, we discovered that the export of lariat intermediates requires Mlp1, a factor implicated in retaining unspliced pre‐‐mRNA in the nucleus of budding yeast. Importantly, Mlp1 promoted the export of lariat intermediates through its interaction with Nab2. Together, our findings establish the importance of the mRNA export pathway in exporting discarded splicing intermediates and implicate a dual role for Mlp1 in not only retaining unspliced transcripts but also exporting partially spliced transcripts. We propose that this dual role of Mlp1 reflects a function in docking mRNAs at the nuclear basket during export.

Support or Funding Information

NIH
Engineering of an Immunogenic pre‐Trans‐splicing RNA (iPTR) to Block Growth and Express a Glioblastoma Specific Epitope
Authors: Falotico Sarah C. , Sivetz Nicole , Nekrasov Peter , Hicks Martin J.
Published: November 2023

The most common and lethal malignancy of the central nervous system (CNS) is glioblastoma multiforme (GBM). Due to the blood brain barrier and the relatively immunologically privileged status of the CNS, clinical strategies have not improved the standard of care. Epidermal growth factor receptor (EGFR), a type of tyrosine kinase receptor, has been found to be overexpressed in as much as 60% of GBM tumors. Upon binding of its cognate ligand, EGFR promotes tumor growth and proliferation. The cytokine, interleukin‐13 receptor alpha variant 2 (IL13Rα2) is an isoform selectively expressed in GBM. It is highly immunogenic, attracting cytotoxic T‐cells to the tumor microenvironment. Therapeutically, this cytokine has the potential reactivate the immune system toward GBM. In the current study, we have designed and cloned an immunogenic pre‐trans splicing RNA molecule (iPTR) against EGFR. The iPTR has the potential to synergistically block growth and reactivate the immune system toward the tumor microenvironment by generation of a GBM‐specific epitope. In a GBM tissue culture model, we use RT‐PCR to detect changes in IL13Rα2 peptide expression. In addition, we use western blot and ELISA to measure changes in EGFR protein localization. Genetic delivery of our highly immunogenic IL13Rα2 peptide using the iPTR has the potential to redirect the immune system to recognize and induce apoptosis in GBM cells.

Support or Funding Information

Monmouth University School of Science Research, Bristol‐Meyers Squibb, Johnson and Johnson, and the Independent College Fund of New Jersey
A Genetic Interaction Between DED1 and HAT1 in Saccharomyces cerevisiae Suggests a Role for Hat1p in mRNA storage
Authors: Hilliker Angie , Kindsfather Audrey , Winters Aidan , Rothbard Nicholas , Robins Sean , Fronek Lisa
Published: November 2023

Ded1p is an essential translation initiation factor that interacts with and is modulated by eIF4F. Ded1p also promotes the assembly and disassembly of stress granules, which contain repressed mRNAs and translation initiation factors. As Ded1p affects both mRNA storage and translation initiation, regulation of Ded1p's function may affect whether mRNAs are stored or translated. To identify regulators of Ded1p in Saccharomyces cerevisiae, we screened for over‐expression suppressors of the severe growth defect conferred by high levels of Ded1p. We found that over‐expression of HAT1, a lysine acetyltransferase that acetylates free histone H4, can suppress this DED1 growth defect. Mutations within the catalytic core of Hat1p substantially reduce this suppression, suggesting that acetyltransferase activity is required for the genetic interaction. We demonstrate that HAT1 antagonizes the accumulation of P‐bodies under short‐term stresses. Under sustained glucose deprivation during diauxic shift and stationary phase, strains lacking HAT1 form more stress granules and have a survival advantage, revealing a new function of Hat1p. Given this genetic connection between HAT1 and DED1, we screened for other lysine acetyltransferases and deacetylases that have a genetic interaction with DED1, identifying several more of these post‐translational modifiers as possible regulators of mRNA storage and/or translation. These results demonstrate new connections between lysine acetylation and the control of cytoplasmic mRNA localization.

Support or Funding Information

Jeffress Memorial Trust; University of Richmond
PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.
Authors: Rothblum Lawrence , Chang Eugenie , Rothblum Katrina
Published: November 2023

When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factors

While S. cerevisae A49, the homologue of PAF53, is not essential for viability, deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25C. This included the genes encoding Rrn3, PAF49 and PAF53. Experiments described by Wang et al. identified PAF53 as a gene “essential for optimal proliferation “. However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology.

Methods

We designed several small guide RNAs (sgRNA) for human PAF53 and cloned them into lentiCRISPR v2. Human 293 cells were transfected with the viral DNA. In some experiments, the cells were cotransfected in parallel with plasmids encoding tagged forms of mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the “CRISPRd” exon, cloned and sequenced to identify mutated PAF53 genes.

Results

We obtained cell lines in which the endogenous PAF53 gene was “knocked out” only when we rescued with FLAF‐PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames.

Conclusions

PAF53 is an essential protein. We did not obtain any clones that did not express the protein.

PAF53 expression requires both genes. We never observed a clone in which one gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not.
Role of cystines in the structure and function of the RNA‐binding protein LARP6
Authors: Peña Eleuterio L , Lane LaPatience F , Salas Eliseo , Castro Jose Miguel , Lewis Karen A.
Published: November 2023

La‐related protein 6, formerly known as Acheron, is an RNA binding protein associated with the translation of mRNAs that encode type‐I collagen. This protein belongs to a superfamily of RNA‐binding proteins known as the La‐related proteins, which share a conserved La Module consisting of a La‐Motif (LaM) and RNA Recognition Motif (RRM). Although extensive research on the mechanism of binding by LARP6 is being performed, very little is known about the physiological conditions that induce the protein to bind to its ligand. A multiple sequence alignment of the LARP6 proteins from vertebrates identified a fully conserved cysteine near the RRM (position C258 in the human protein), and two semi‐conserved cysteines near the C‐terminal region (positions C378 and C490 in human LARP6). To determine if disulfide bonds were present in the protein, the reducing agent dithiothreitol (DTT), was added during purification. In the presence of DTT, LARP6 eluted from a size exclusion column at a volume that is indicative of an increase in the hydrodynamic radius of the protein. Therefore, we hypothesize that at least one pair of the conserved cysteine residues is involved in intramolecular disulfide bonding. Using the human LARP6 (HsLARP6) as a model, the location of the structurally relevant disulfide bonds is being identified using a combination of site‐directed mutagenesis, chemical modification, and mass spectrometry. By mutating each cysteine to serine, we can monitor changes in protein stability through limited proteolysis and chemical and thermal denaturation assays. Additionally, the effects of cysteine mutagenesis on the biochemical activity of LARP6 will be assessed using electrophoretic mobility shift assays against the previously identified stem‐loop structure in the 5′UTR of the COL1a1 mRNA. The identification of the existence and function of disulfide bonds within LARP6 will provide further understanding of the physiological function of this posttranscriptional regulator.

Support or Funding Information

Funding provided by the Texas State University Department of Chemistry and Biochemistry; the National Science Foundation through the Texas State University CheMIE Summer Research Experience for Undergraduates (NSF 1156579 to A.K.), the National Institutes of Health through the South Texas Doctoral Bridge Program (GM102783 to J.M.C., E.S. & ELP), the Student Undergraduate Research Fund through the Texas State University Honors College (to E.L.P.), and the National Institutes of Health grant (GM119096 to K.A.L).
Effect of Histidine Rich Sequence Motifs on the RNA Binding Activity of LARP6
Authors: Toner Chelsea , Betancourt Francisco C. , Lewis Karen
Published: November 2023

La Related Protein 6 (LARP6) is a sub‐family of RNA binding proteins that are members of from the superfamily of La‐Related Proteins (LARPs). In vertebrates, LARP6 is known to bind to the 5′ untranslated region of mRNAs that encode for type I collagens. In contrast, the function of the three LARP6 paralogs in plant LARP6 (LARP6a, LARP6b and LARP6c) is much less characterized. Of the three variations of the LARP6 protein in Arabidopsis thaliana, LARP6a and LARP6c have been shown to bind to poly‐adenosine, poly‐uracil and poly‐cytosine RNA oligonucleotides. This ability to bind a variety of RNA sequences may indicate that plant LARP6 proteins act as translocators, moving mRNAs from the nucleus to the cytoplasm. Unlike LARP6a and LARP6c, LARP6b contains an endogenous histidine‐rich (His‐rich) sequence. His‐rich sequences play diverse roles in biology, including creating a favorable electrostatic surface for intermolecular interactions, directing subcellular localization of proteins, and binding to divalent heavy metals. We hypothesize that the His‐rich sequence in LARP6b may contribute to the RNA binding activity of the protein. Mutant forms of the Arabidopsis LARP6 proteins were constructed using site‐directed mutagenesis, and were expressed and purified from E. coli. Protein structure was analyzed by size exclusion chromatography and limited proteolysis, which provided insight about the structural contributions of the His‐rich sequences. RNA binding assays were carried out with a variety of putative ligands, and demonstrated the effects of deleting the His‐rich sequences in LARP6b. RNA binding was also performed in the presence of select heavy metals known to tightly interact with His‐rich sequences, including Ni2+ and Co2+. These studies provide novel insight into how plants have adapted His‐rich sequences for function in the La‐related protein family of RNA‐binding proteins.
Structural probing of the cap‐independent vascular endothelial growth factor messenger RNA
Authors: Huang Wei , Scott Harry , Merrick William , Bhattacharyya Debmalya , Basu Soumitra , Taylor Derk
Published: November 2023

Internal ribosome entry sites (IRES) are cis‐regulatory elements located at the 5′ leader region of certain messenger RNA transcripts. IRESs have the capacity to recruit the translation machinery to initiate protein production in a cap‐independent mechanism. Structural studies on viral IRESs have articulated a structure based mechanism that viral IRES RNAs adopt secondary structures that are favorable for ribosome binding and place mRNAs in the decoding center to promote initiation of protein synthesis. Despite ~10% of eukaryotic mRNAs possess 5′ IRESs, structural information towards functional elucidation of cellular IRESs are still lacking. Cellular IRESs have showcased their importance particularly under stress conditions when cap‐independent protein synthesis is not impaired as cap‐dependent translation is. Therefore, at least under certain conditions, cellular IRESs provide a mechanism of translational control for a select set of proteins. An important and well‐studied example is that of vascular endothelial growth factor (VEGF), where the 5′ untranslated region of the mRNA behaves as an IRES to enhance expression of VEGF during malignant transformation. Here we used chemical probing and cryo‐electron microscopy (cryo‐EM) to investigate the structure of VEGF IRES in solution and when it is bound to the small ribosomal subunit. Integrating experimental information from selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) analysis, we generated a secondary structure model for the VEGF IRES RNA. In addition, we present a 10Å cryo‐EM density map of the VEGF IRES in complex with 40S ribosomal subunit. The structural information enables us to identify the location that the cellular IRES occupies on the 40S ribosomal subunit to mimic an initiation‐like state.
A Cellular Non‐coding RNA Activator of Human 2′‐5′‐Oligoadenylate Synthetase 1
Authors: Calderon Brenda Milagros , Conn Graeme Leslie
Published: November 2023

Human 2′–5′‐oligoadenylate synthetase 1 (OAS1) senses double‐stranded RNA (dsRNA) and is a key component of the innate immune response to viral infection. Binding of dsRNA activates OAS1 and results in synthesis of 2′–5′ linked oligadenylate second messengers that in turn activate ribonuclease L (RNaseL) and lead to cleavage of viral and cellular RNA. The recently discovered cellular non‐coding RNA 886 (nc886) is an endogenous inhibitor of another dsRNA‐sensing innate immune protein, the double‐stranded RNA activated protein kinase (PKR). We tested the hypothesis that nc886 might also act as a regulator of OAS1 and found, surprisingly, that nc886 potently activates OAS1, rather than inhibiting its activity. nc886 RNA adopts two structurally distinct conformers. OAS1 functional assays combined with RNA structure probing demonstrate that potent OAS1 activation is dependent on a complex RNA structure present within the apical region of only one nc886 conformer. Deletion or sequence modification of the apical stem‐loop or an adjacent internal asymmetric loop results in near complete loss of activity. In contrast, truncation of nc886 at its base‐paired termini results in minimal loss of activity. Furthermore, mutation of two known determinants of OAS1 activation, a consensus sequence and a 3′‐single‐stranded pyrimidine motif, present in nc886 result in minimal loss of activity. Our findings demonstrate that the degree of OAS1 activation is not solely dependent on dsRNA length; rather, potent OAS1 activity can be induced by small structured RNA, such as that present in nc886 RNA. Additionally, this is the first demonstration of a potential endogenous RNA regulator of OAS1 and suggests a mechanism for amplifying the immune response during infection.
Breaking barrier integrity in alveolar epithelial cells leads to modification in the expression profile of tight junction and ion transporter genes
Authors: Dagenais André , Desjardins Julie , Berthiaume Yves
Published: November 2023

Acute lung injury is associated with epithelial disruption and abnormal transepithelial Na+ transport affecting lung oedema clearance and injury recovery. In the past, we reported that ENaC expression is downregulated in alveolar epithelial cells in presence of pro‐inflammatory conditions. However, the impact of loss of barrier integrity on ion channels and genes important for barrier integrity in these cells is not known. In the present work, we determined if loss of barrier integrity, in absence of inflammation, would have an impact on the gene expression profile of rat alveolar epithelial cells in primary culture, focussing on ion transporter and tight junction genes. We found that mechanical wounding (MW), induces a ~60% decrease of α,β and γENaC mRNA expression at 6h and 24h and ~40% decrease of αNa/K‐ATPase mRNA expression. ENaC short‐circuit current (Isc) recorded in Ussing chamber was decreased by 80% at 6h following MW and was correlated to αENaC mRNA expression (R=0,8379). Beside ENaC and Na/K‐ATPase, the expression profile of genes important for restauration of barrier integrity or epithelial differentiation was investigated at 6h following MW. There was an upregulation of tight junction genes (Cldn3 (171%), Cldn4 (288%), but not of Cldn5 and Cldn18. Integrin (Itgb3 (160%)) and epithelial markers (Ker8 (153%), Ker18 (209%)) were upregulated at 6h along with Ker14 (279%). While Cldn 3 and Cldn4 expressions were back to normal after 24h, αENaC gene expression was normalized only after 48h. This expression profile was also observed if we disrupt tight junction with a short EDTA treatment. Altogether the present data suggest that breaking epithelial barrier integrity leads to a change in epithelial cells phenotype where the cells prepare themselves to repair and restore tight junctions and cell polarity. Our results show that disruption of barrier integrity also affects ENaC expression and activity. These data will help to better understand the consequences of alveolar epithelial injury in the resolution of edema following lung injury.
Sphingosine Kinase‐1 inhibitor, PF‐543 ameliorates Bronchopulmonary Dysplasia in a neonatal murine model
Authors: Harijith Anantha , Ha Alison W , Ebenezer David L , Fu Panfeng , Berdyshev Evgeny , Kanteti Prasad , Natarajan Viswanathan
Published: November 2023

Background

We have earlier demonstrated that genetic knock out of sphingosine kinase1 (SphK1) ameliorated hyperoxia (75%) induced lung injury in a neonatal murine model of bronchopulmonary dysplasia (BPD). SphK 1 catalyzes the formation of sphingosine‐1‐phosphate (S1P), which acts through G protein coupled receptors S1P1–5. Exposure of neonatal mice pups to hyperoxia enhanced SphK1 expression and S1P levels and promoted formation of reactive oxygen species (ROS) in lungs. In vitro, exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia (3–24 h) stimulated SphK1 activity, S1P generation and ROS production via p47phox, which was blocked by PF‐543, a potent and specific SphK1 inhibitor.

Hypothesis

PF‐543 protects neonatal lungs from hyperoxia‐induced BPD.

Methods

Neonatal mice were administered PF‐543 (5 mg/kg/dose, intraperitoneal injection) starting on postnatal day 3 (PN3) and exposed to normoxia or hyperoxia (75% O2) for 7 days. Mice received three additional doses of PF‐543, which were administered on alternate days and mice were sacrificed on PN 11. Broncho alveolar lavage (BAL) fluid and lung tissue were collected, and markers of inflammation and lung histology were evaluated. Plasma S1P levels were measured to determine the efficacy of PF‐543 in inhibiting SphK1.

Results

SphK1 inhibition by PF‐543 resulted in amelioration of hyperoxia‐induced BPD as evidenced by improved alveolarization characterized by decreased mean linear intercept compared to controls. Also, neonatal pups that received PF‐543 showed decreased infiltration of inflammatory cells in alveolar space, lower levels of protein and ROS in BAL fluid compared to controls. Administration of PF543 was accompanied by reduced plasma S1P levels.

Conclusions

Inhibition of SphK1 activity by, PF‐543 significantly reduced hyperoxia‐induced lung inflammation and BPD in newborn mice. These results suggest that PF‐543 can serve as a potential therapeutic drug to treat BPD.

Support or Funding Information

This work was supported by the University of Illinois at Chicago (UIC) Proof of Concept grant to A.H and by the National Institutes of Health Grant P01 HL98050 to V.N.
Sphingosine‐1‐phosphate as an angiocrine mediator in type II cell mediated alveoli epithelial repair following lung injury
Authors: Chen Qian , Huang Longshuang , Fu Panfeng , Rehman Jalees , Malik Asrar , Natarajan Viswanathan , Liu Yuru
Published: November 2023

The alveolar barrier is continuously exposed to pathogens and is highly susceptible to inflammation and injury. Hence, orderly and exquisitely regulated repair of injured alveoli is essential, but the underlying mechanisms are not fully understood. Lung alveolar epithelium is composed of Type I and Type II cells. While Type II cells occupy only 5% of the surface area, they act as progenitor cells by self‐renewal and differentiation into Type I cells to repair the injured alveolar barrier. Type I cells occupy 95% of the surface area and enable gas exchange and are juxtaposed to the lung microvascular endothelium. Lung microvascular ECs comprising up to 40% of the lung cells, may play a major (but as yet unappreciated) role in influencing the regeneration of alveolar epithelium. Using genetically modified mice in which generation of Sphingosine‐1‐phosphate (S1P) specifically in endothelial cells was blocked [(through deletion of sphingosine kinase 1 (SphK1)], we have found evidence that endothelial‐derived S1P participated in repairing of the alveolar epithelium. After acute lung injury were induced in these mutant mice by i.t. Pseudomonas aeruginosa, Type II cells were abnormal in that they showed higher proliferation and these cells failed to show reduced expression of the Type II cell marker Sp‐C during the phase of Type II to Type I transition, suggesting a failure to differentiate into Type I cells. We also found the expression of S1P receptor S1PR2, which is a G protein‐coupled receptor, was induced on Type II cells post injury and preceding alveolar repair. Our results indicate that lung microvessel ECs release S1P after injury and activate receptor S1PR2 on Type II alveolar epithelial cells to initiate Type II to Type I cell transition required for alveolar repair.

Support or Funding Information

NIH National Heart, Lung, and Blood Institute Grant R01HL105947.
IL‐6 modulates cardiac muscle proteostasis and anti‐oxidative capacity post‐burn
Authors: El Ayadi Amina , Wang Ye , Prasai Anesh , Wetzel Michael , Goullry Ashley , Porter Craig , Herndon David N , Finnerty Celeste
Published: November 2023

Heart failure is the number one cause of death in severely burned patients. Burn‐induced cardiac dysfunction includes increased cardiac work, tachycardia and increased energy expenditure. Burn injury induces a surge in catecholamine and cytokines levels that lasts up to 3 years post injury. While cardiac dysfunction has been associated with stimulation of the β‐adrenergic receptors (β‐ARs) by catecholamines, the role of inflammatory cytokines in the cardiac muscle function is still unclear. We have reported that burn injury alters the beta‐adrenergic signaling in the heart at 7 days post‐burn. In another study, we have shown that burn injury induces atrophy of the diaphragm muscle through activation of the IL‐6/JAK/Stat pathway.

Male wild‐type and interleukin 6 knockout mice (IL‐6 KO) were subjected to a 30% total body surface area full‐thickness burn. Animals were sacrificed 10 days post injury. The hearts were isolated and western blotting performed to analyze the role of JAK/Stat signaling pathways and antioxidant enzymes in cardiac muscle proteostasis post‐burn.

Burn injury significantly increases Stat‐3 phosphorylation at Ser 727 in the WT (p<0.05) but not in the IL‐6‐KO animals. Stat‐3 phosphorylation at tyrosine705 (Y705) was activated in the cardiac muscle of both the WT (p<0.05) and IL‐6 KO (p<0.05) mice post‐burn. At basal levels, p‐Stat‐3(Y705) was higher in the control IL‐6 KO than the control WT animals (p< 0.05). P‐Stat‐3 (Y705) levels were also higher in L‐6 KO burned animals compared to WT burned animals (p<0.01). The protein levels of antioxidant enzymes SOD‐2 and catalase were significantly decreased post‐burn in the WT (both p<0.05) but not in the IL‐6 KO animals.

These data suggest that the molecular mechanisms regulating cardiac muscle proteostasis and anti‐oxidative capacity following burn injury are altered in the IL‐6‐KO mice supporting the role of IL‐6 as a critical mediator in these processes.

Support or Funding Information

This study was supported by grants from the National Institute of Health (P50‐GM60338, R01‐GM56687, and T32‐GM8256, NIH R01 GM 112936‐01, UL1TR000071), Shriners Hospitals for Children (71008, 71001 and 84080), and the Shriners Hospital for Children fellowship for AE (SHC 84202).
Characterization of a Short Motif Capable of Enhancing Human Cap‐Independent Translation
Authors: Juba Amber N. , Hansen Allison , Chaput John C. , Wellensiek Brian P.
Published: November 2023

The classical model for eukaryotic translation initiation involves linear scanning, a cap‐dependent process in which the ribosome recognizes a 7‐methylguanosinecap at the 5′ end of the RNA message and scans the 5′ untranslated region for an appropriate initiation site. Alternatively, a growing body of evidence suggests that cap‐independent mechanisms play an important role during many cellular events where cap‐dependent scanning is inhibited. However despite this evidence, the mechanism by which this cap‐independent mode of translation occurs remains largely unknown. In a previous study, we began to address this by combining messenger RNA display with next‐generation deep sequencing to profile the human genome for RNA leader sequences that can enhance the translation of uncapped mRNA transcripts. Analysis of these sequences revealeda short, 13 nucleotide motif that was present in many of the RNAs isolated via this method. Given these findings, we propose that the identified 13 nucleotidemotif, when present within the leader sequence of an open reading frame, has an integral role in determining the increased translational activity driven by the larger leader sequence. To explore this role we examined the translation enhancing activity of several human in vitro selected translation enhancing elements (TEEs), which either contained or did not contain the 13 nucleotide motif. A series of reporter vectors containing the TEEs were used to assay the effect of the presence and absence of the motif on overall translation efficiency. Functional analysis studies performed in vitro and in cultured human and mammalian cells indicate that the 13 nucleotide motif, when present within a larger TEE, increased protein translation levels by several fold. Additional studies have identified distance constraints for translation enhancement with respect to the 13 nucleotide motif and the downstream open reading frame. Taken together these results provide evidence that the identified short 13 nucleotide motif plays an important role in the mechanism of cap‐independent translation from the human genome. This observation provides a new opportunity to explore the mechanistic details of human translation enhancing elements and their contribution to the human proteome.

Support or Funding Information

Funding for this research was provided by Midwestern University intramural funds awarded to BPW, a Midwestern University College of Veterinary Medicine research fellowship awarded to AH, and an NIH grant awarded to JCC