bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrating Human Cell Culture into Undergraduate Research Projects
Authors: Taylor Christopher Edward
Published: November 2023

One of the most important aspects of undergraduate research is to prepare students with the conceptual knowledge and technical skills that they will need for graduate or industrial research. Projects that involve human cell culture and the biochemical methods used to evaluate cell viability, protein expression, and mRNA production are an effective way to build up both the concepts and technical skills that undergraduates will need. However, these projects also tend to require substantial investments of time and money, which are often difficult at the institutions which typically focus on undergraduates.

This presentation will provide ways to adapt cell culture based projects to the resources – particularly funding, student time/expertise, and advisor time – that are realistically available at a primarily undergraduate institution. Aspects of this discussion will include: maintaining cells and running plate based experiments given relatively inexperienced undergraduate researchers, improving undergraduate familiarity with topics that typically go beyond the undergraduate curricular offerings, minimalist budgeting for cell culture based projects, and managing several undergraduate research projects on a typical PUI teaching load.
Effect of Mitoquinone Treatment on Cardiac Function and Pathophysiology in Pressure Overload‐induced Heart Failure
Authors: Goh Kah Yong , Wende Adam R , Soorappan Rajasekaran N. , Halade Ganesh , Darley‐Usmar Victor , Jinno Miki , Prabhu Sumanth D. , Zhou Lufang
Published: November 2023

Mitochondrial dysfunction has been implicated in the pathogenesis of heart failure (HF). In particular, emerging studies have indicated that mitochondrial‐derived ROS are involved in calcium dysregulation, ventricular remodeling, cardiac cell death, and metabolic abnormality in failing hearts. Mitoquinone (MitoQ), an antioxidant specifically targeted to mitochondria, has been repeatedly shown beneficial effects in mitochondrial‐related diseases. However, the protective effect of MitoQ in pressure overload HF has not been investigated. We hypothesized that MitoQ treatment improves cardiac function and mitigates pathological remodeling by attenuating mitochondrial‐derived oxidative stress. To test the hypothesis, 8‐week male mice were subject to ascending aortic constriction (AAC) to induce acute pressure overload HF. Cardiac function was assessed with echocardiography. Gravimetric data were collected at the end of the studies. Hematoxylin and eosin (H&E) and picrosirius red (PSR) staining was used to assess the histologic characteristics and TUNEL assay was used to evaluate cardiac cell death. Results showed that one week MitoQ treatment improved fractional shortening and ejection function and reduced the heart mass to tibia length ratio in the AAC mice. In addition, collagen deposition in AAC mice were attenuated by MitoQ treatment. Together, these findings suggest that MitoQ treatment confer protective effect in pressure overload‐induced heart failure in mice.
Quantitative Multi‐Parametric Microscopy Analysis of Live 3D Cell Models
Authors: Dmitriev Ruslan I. , Jenkins James , Okkelman Irina A. , O'Donnell Neil
Published: November 2023

Three‐dimensional (3D) cell models are at the forefront of cancer cell biology and tissue engineering, providing opportunity for better in vitro mimicking of cell morphology, membrane transport, physiological O2, pH and metabolite diffusion gradients, lacked in conventional cell culture. However, studying the biochemistry of the cell in 3D demands new methods of quantitative fluorescence microscopy such as fluorescence and phosphorescence lifetime imaging microscopies (FLIM, PLIM). Here we report development and application of imaging protocols for quantitative microscopy of molecular oxygen (O2) and temperature (T), combined with analysis of cell death in tumor spheroids from human colon carcinoma HCT116 [1, 2]. By analyzing gradients of pH, O2 and fluorescence‐based viability markers (CellTox Green, TMRM) we optimized the method of spheroid formation, providing best overall cell viability and reproducibility. By measuring T and O2 in spheroid models, we identified T as new metabolic marker, dependent on the 3D organization of cell [2]. We also thoroughly evaluated the available and introduced a number of novel nanoparticle‐based probes for high‐resolution imaging of hypoxia (O2), pH and T by FLIM method. Overall, we see the described methodology as enabling quantitative microscopy of advanced 3D cell models in tissue engineering and regenerative medicine.

Support or Funding Information

Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.
Potent DNA Strand Annealing Mediated by the T7 Single‐Stranded DNA Binding Protein gp2.5
Authors: Hernandez Alfredo J. , Richardson Charles C.
Published: November 2023

Single‐stranded DNA binding proteins are ubiquitous in all domains of life and play essential roles in DNA replication, recombination, and repair. Bacteriophage T7 encodes a single‐stranded DNA binding protein, gp2.5, which is crucial for coordinating the synthesis of leading and lagging DNA strands.

In addition to its role in DNA replication, T7 gp2.5 displays a potent homologous DNA strand annealing activity, which is necessary for genetic recombination. Gp2.5 enhances the annealing of DNA strands orders of magnitude over the spontaneous rate, and is considerably more efficient compared to other recombination proteins. The annealing of complimentary strands by gp2.5 is strictly specific for DNA. While the annealing reaction is somewhat tolerant to internal mismatches, it is very permissive to internal, unpaired loops.

Here we provide a model for the mechanism of gp2.5‐mediated homologous strand annealing and explore its applicability in oligonucleotide‐mediated gene modification and the formation of higher‐order DNA structures in vitro.
Y‐family DNA Polymerase Human Pol Kappa Is More Tolerant of Changes to Its Active Site Loop Than DinB
Authors: Antczak Nicole M , Packer Morgan , Walsh Jason , Ippoliti Paul , Beuning Penny J
Published: November 2023

DNA damage is a constant threat from both endogenous and exogenous sources. Exposure to UV radiation and reactive oxygen species can cause DNA damage. Most damage undergoes a repair process but damage that is not repaired is bypassed in a process called translesion synthesis. Y‐family DNA polymerases carry out translesion synthesis by replicating damaged DNA as they have a larger active site to accommodate bulky DNA adducts. Y‐family DNA polymerases are conserved in all domains of life. In E. coli, there are two, DinB (Pol IV) and UmuD′2 (Pol V), one in the archaeon Sulfolobus solfataricus, Dpo4, and several in humans including pol kappa, an ortholog of DinB. Both DinB and pol kappa have been shown to bypass minor groove adducts on the N2 position of deoxyguanine while Dpo4 can bypass both minor groove and major groove adducts. Although DinB and pol kappa are homologous, we have previously shown they are blocked to different extents by major groove lesions etheno‐dA and N6‐furfuryl‐dA and that a single point mutation in an active site loop in DinB eliminates discrimination against N6‐furfuryl‐dA.

In this work we created chimeric polymerases by swapping the loop region adjacent to the active site using site directed mutagenesis to assess the importance of the loop in damage specificity and activity. Using primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adducts etheno‐dA and N6‐furfuryl‐dA, as well as undamaged templates, we found the loop regions of DinB and pol kappa near the catalytic site are important for damage bypass specificity and accuracy of nucleotide incorporation. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of major groove adducts by DinB and hPol κ can be accomplished.

Support or Funding Information

Support from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.
Role of Pol κ and DinB distal residues on extension step of TLS
Authors: Stern Hannah R , Mills Caitlyn L , Ondrechen Mary Jo , Beuning Penny J
Published: November 2023

DNA is subject to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass lesions when copying damaged DNA in a process called translesion synthesis. DinB is one of these polymerases, which is involved in bypassing deoxyguanosine adducts at the N2 position. There are also four human Y family polymerases, including DNA polymerase Kappa, that have similar function. However, Pol κ is more efficient in copying past DNA damage lesions in the extension step of translesion synthesis. In order to investigate the importance of particular residues in the extension step of TLS, the computational method POOL was utilized. This tool identified active site residues and residues previously observed to be important for activity. POOL also predicted more distant residues that do not have direct contact with substrate that may have catalytic importance, but the residues are in different regions of DinB and Pol κ. In addition, previous HXMS work done in our lab suggested a possible allosteric change in both DinB and Pol κ in different distal residues that POOL did not predict. To study the contribution of these distal residues on the extension step of TLS, DinB and Pol κ variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage lesions.

Support or Funding Information

Support from NSF‐MCB‐1517290, American Cancer Society RSG‐12‐161‐01‐DMC, and the PhRMA Foundation (predoctoral fellowship in informatics awarded to CLM)
Investigating the mechanism of trans‐lesion synthesis by human DNA polymerase kappa
Authors: Allen Taylor Brooke , Younger Zane , Benitez Benedetta Sampoli
Published: November 2023

Our DNA is constantly under attack by both exogenous and endogenous chemicals that have the ability to damage it. If not repaired, these DNA adducts can lead to mutations to the DNA or even worse, they can halt replication, eventually leading to aging and/or cancer. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases, known for their ability to conduct trans‐lesion synthesis of damaged DNA. So far, the mechanism for lesion bypass discrimination by pol k is unclear. Recent studies suggested that similar to other polymerases, pol k might cycle between different open and closed conformations upon binding to DNA and the substrate. Through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotides. In particular, we simulated a binary system, with DNA and protein only, ternary systems with correct and incorrect incoming nucleotides, and a ternary system with with a O6‐methylated guanine (O‐metG) on the templating strand, a damage which pol k cannot bypass. Preliminary results show that pol k does not attain a close, catalytically competent state both in the binary and the O‐metG system, as evidenced by the few contacts between the protein and the incoming nucleotide. We have also identified key residues important for the stabilization of the incoming nucleotide as well as a hypothesis to why the O‐metG does not get bypassed as efficiently as other damages.

Support or Funding Information

This work was funded by the Rose M. Badgeley Residuary Charitable Trust and the Sister Colette Mahoney Science Research Grant.
Interactions between variant histone H2A.Z and linker histone H1 in budding yeast
Authors: Riggs Julianne , Huang Junning , Winston Lisle , Sica Margauex , Holmes Scott
Published: November 2023

Loss of the highly conserved histone variant H2A.Z, encoded by HTZ1, in budding yeast leads to alterations in genomic integrity, including improper establishment of silencing and defects in chromosome cohesion. We have shown that deletion of HHO1, the gene encoding linker histone H1, suppresses the silencing and plasmid retention defects observed in Δhtz1 cells. HTZ1 is synthetically lethal or sick with several dozen yeast genes of varying function. To examine the specificity of the H1‐H2A.Z interaction, we performed a synthetic screen to see if deletion of HHO1 could rescue cell viability or growth in Δhtz1. Our data suggests that loss of H1 can suppress synthetic defects and that this suppression is specific for strains lacking proteins involved in histone deacetylation or ubiquitination. Finally, we have used chromatin immunoprecipitation to establish that strains lacking H2A.Z show reduced association of histone H1 with chromatin. Similarly, strains lacking H1 show reduced association of histone H2A.Z with chromatin. Overall our experiments suggest that loss of H2A.Z triggers an H1‐dependent disruption of normal chromatin structure.
INO80 Chromatin Remodeling Connects Metabolic Gene Expression to Cell Division
Authors: Morrison Ashby J , Gowans Graeme , Schep Alicia , King Devin , Greenleaf William
Published: November 2023

The coordination of cellular function with the environment is essential for adaptation and survival. S. cerevisiae has a remarkable ability to sense diverse (i.e. nutrient‐rich or □limiting) environments and reprogram their energy metabolism and proliferative capacity accordingly. These adaptive cellular responses are often achieved by rapid inducible changes in metabolic gene expression facilitated by chromatin modification.

Previously we identified the INO80 chromatin remodeler as a primary regulator of gene expression in energy metabolism pathways (Yao et al, MCB 2016). We then sought to examine the role of INO80 in coordinating metabolic homeostasis with cell division in metabolically synchronized yeast. Synchronization is achieved through nutrient depletion of yeast cultures followed by constant perfusion of glucose. Subsequently, the Yeast Metabolic Cycle (YMC) is induced, characterized by continuous respiration cycles of oxygen consumption. Within the YMC, most genes are periodically expressed in specific phases of quiescence, cell growth, and cell division. However, little is known about how these genes are so precisely regulated.

We find that the INO80 complex is critical for organizing global gene expression and chromatin organization in the YMC. Resulting cellular phenotypes include altered respiration cycles and loss of metabolic coordination with cell division. Thus, the INO80 chromatin‐remodeler is central regulator of connecting metabolic gene expression to cell division.

Support or Funding Information

National Institutes of Health; R35 GM119580
Biochemical Insights into the Mechanism of Oncohistones
Authors: Lewis Peter W
Published: November 2023

Recently, genome‐wide sequencing technologies have allowed the notable discovery of somatic mutations in chromatin modifying factors in many types of cancers. The collective number of oncogenic mutations has led to the emerging view of “driver mutations” in chromatin regulators underlying many human malignancies. Specific pediatric brain and bone tumors are reported to harbor a remarkably high frequency of monoallelic, gain‐of‐function mutations in genes encoding canonical (H3.1/2) or variant (H3.3) histone H3. These high frequency mutant histones (collectively referred to as ‘oncohistones’) are suspected drivers of specific types of human cancer. Previously, we demonstrated that lysine‐to‐methionine (K‐to‐M) substitutions transform histone H3 proteins from substrates into potent and highly specific inhibitors of SET domain‐containing lysine methyltransferase enzymes (e.g. K27M found in ~80% of midline gliomas, and K36M found in ~95% of chondroblastomas) and affect overall levels of histone modifications. We find that that the cofactor S‐adenosyl methionine (SAM) is necessary for stable interaction between SET domain enzymes and K‐to‐M oncohistones. Consistent with the formation of a ternary complex, we find that oncohistones are uncompetitive inhibitors with regard to SAM.

Given the restricted distribution of H3 mutations in human cancers, we hypothesize that cell lineage is crucial for the ability of oncohistones to promote tumorigenesis. Therefore, we are using a combination of biochemical investigation, accurate model generation, and study of human tumor samples to comprehensively understand how H3 mutations mediate tumorigenesis. We find that the H3 K36M mutant histone impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. Depleting H3K36 methyltransferases, or expressing an H3 K36I mutant that similarly inhibits H3K36 methyltransferases, is sufficient to phenocopy the H3 K36M mutation. Upon loss of H3K36 methylation, a genome‐wide gain in H3K27 methylation leads to a redistribution of PRC1 and de‐repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified. I will discuss our recent work to characterize how oncohistones perturb cellular differentiation through inhibition of specific chromatin‐modifying enzymes.
Free hemoglobin change gene expression involving in cell‐cell signaling through different DNA methylation in THP‐1‐derived macrophages
Authors: Cha Min Ho , Lee Myeong Soo
Published: November 2023

Background

Free hemoglobin formed from hemolysis of red blood cell, was known a predicator of many disease such as cancer, sclerosis and delirium. That also related with cytokine production in leukocytes. In this study, we elucidated change of gene expression through DNA methylation difference by free hemoglobin in THP‐1 derived macrophage.

Methods

PMA induced THP‐1 derived macrophage were treated 50ug/ml of free hemoglobin (purchased from Sigma). DNA methylation were analyzed through whole genome bisulfate sequencing (WGBS) using TruSeq® DNA Methylation Kit (illumina) and 을 Illumina HiSeq. platform (illumina). Sequence quality were checked by FastQC and Trimmomatic and data analyzed by BSMAP. Difference of DNA methylation were determined in delta‐mean>[0.75] and p <0.05). Expression of genes were determined by reverse transcriptional PCR (RT‐QCR) and q‐PCR.

Results

DNA methylation of 29,706 CpG sites in total genome and 553 CpG sites in promoter region were changed by hemoglobin. Among 553 CpG sites in promoters, 267 CpG sites were hypomethylated and 286 sites were hypermethylated. GO annotation showed genes involving in neurological system process, ion transport, cell‐cell signaling and skeletal system development were significantly affected by hemoglobin. Genes involved in p53 cardiovascular system, cell adhesion molecules were significantly affected. Among of them, we focused 23 genes, which were included in cell‐cell signaling, and checked gene expression of five genes. IL‐7 and PCDN1 showed significant change of gene expression.

Discussion

Our results showed that increase of free hemoglobin affects gene expression through change of DNA methylation and these might suggest that free hemoglobin level in vessel should be affect cell‐cell signaling and free hemoglobin might predictor of some diseases.

Support or Funding Information

This research was supported by a Grant from the Korea Institute of Oriental Medicine (C16111) and funds from Korea Food Research Institute (G16020)
Upregulating Human Leukocyte Antigen Expression in Multiple Cancer Cells through Epigenetic Modulators
Authors: Terrigino Nicholas Taylor , Nicholas David Joseph , Powers Rachel Elizabeth , Dutta Shourik , O'Donnell Robert Watts
Published: November 2023

This study attempts to discern whether two epigenetic modulators, 5‐Azacytidine and Entinostat, increase the expression of Human Leukocyte Antigen (HLA) proteins on human cancer cells when used in combination. HLA is routinely down‐regulated in cancer, presumably to escape immunologic detection. We have previously shown that 5‐Azacytidine, a cytosine DNA methyltransferase inhibitor, increased HLA expression. Others have shown that Entinostat, an epigenetic modulator that functions as a histone deacetylase inhibitor, can modify tumor growth both in vivo and in vitro. Five cancer cell lines, HL‐60, MDA‐MB‐435, MDA‐MB‐231, MCF‐7, and HTB‐4 cells are being tested using flow cytometry and Phycoerythrin (PE) conjugated antibodies against the HLA Class I antigens A, B and C. Initial findings show that Entinostat, and/or the combination of Entinostat and 5‐Azacytidine, are more effective at up‐regulating HLA expression than 5‐Azacytidine alone in HL‐60, HTB‐4, and MDA‐MB‐435 cell lines. Further cells lines will be tested to determine if Entinostat or the combination will be more effective in up‐regulating HLA expression as well as the optimal dose and exposure time. If 5‐Azacytidine and Entinostat are shown to work in synergy, cancer cells could become better targets for cancer immunotherapy.

Support or Funding Information

Geneseo Foundation and the SUNY Geneseo Biology Department
A Shared Structural Recognition Element in mRNA Substrates of the tRNA Modifying Enzyme Pseudouridine Synthase 1
Authors: Carlile Thomas M , Bell Tristan A. , Rojas‐Duran Maria F , Zinshteyn Boris , Shin Hakyung , Mason Caitlyn , Gilbert Wendy V
Published: November 2023

RNA is extensively chemically modified in all organisms. These modifications were thought to be restricted to stable non‐coding RNAs, however recent work has revealed the presence of a variety of post‐transcriptional modifications in mRNA. Our lab recently developed Pseudo‐seq, a transcriptome‐wide approach to pseudouridine identification, which led to the discovery of widespread pseudouridylation of mRNAs in yeast, mouse, and human cells. We found that mRNA pseudouridylation events are regulated in response to cellular growth state; however, little is known about the mechanisms responsible for selective, regulated targeting of specific sequences for modification. We have sought to understand the mechanisms underlying how the conserved pseudouridine synthase Pus1 recognizes its mRNA targets. We have reconstituted Pus1‐dependent modification of synthetic mRNAs in vitro. By computational prediction, high‐throughput RNA structure probing, and mutational analysis we have revealed a conserved RNA structure that is necessary and sufficient for mRNA pseudouridylation by Pus1. These results provide insight into how mRNA targets are recognized by pseudouridine synthases, and our experimental approach is applicable to the study of all enzyme‐mRNA interactions.

Support or Funding Information

This work was supported by grants from The American Cancer Society – Robbie Sue Mudd Kidney Cancer Research Scholar Grant (RSG‐13‐396‐01‐RMC) and the National Institutes of Health (GM094303, GM081399) to W.V.G.

T.M.C. was supported by the American Cancer Society New England Division (Ellison Foundation Postdoctoral Fellowship).

This work was supported in part by the NIH Pre‐Doctoral Training Grant T32GM007287.
Dissecting the Mechanism of H3K36 Methylation in Regulating Pre‐mRNA Splicing
Authors: Leung Calvin , Douglass Stephen , Johnson Tracy
Published: November 2023

Eukaryotic genes contain introns that are removed during pre‐mRNA splicing by the spliceosome, a large, dynamic RNA‐protein complex composed of five ribonucleoprotein complexes and many associated protein cofactors. Although the detailed mechanism of spliceosome assembly onto pre‐mRNA has been elucidated using elegant biochemistry, in vivo pre‐mRNA splicing is tightly coordinated with gene transcription. Splicing and transcription appear to be coupled not only in time, but also functionally. Spliceosome assembly and the subsequent catalytic steps take place in the context of a dynamic chromatin environment, which suggests that the state of the chromatin may affect splicing. Nonetheless, the precise role of histone modifications in contributing to splicing have remained unclear. Here, we investigate the role of the histone methyltransferase Set2 and its associated chromatin mark, H3K36 methylation, in determining splicing outcomes. Through high‐throughput RNA‐sequencing, we identify discreet classes of lowly expressed transcripts whose splicing is strongly dependent on an intact histone H3K36 methyltransferase complex. Moreover, this same subset of genes shows defective splicing when the H3K36 residue is mutated to prevent its methylation, reinforcing that it is histone methylation per se that is important for splicing these introns. To understand the mechanism by which H3K36 affects splicing, we have analyzed molecular events downstream of H3 methylation, and have identified the NuA4 complex and its chromodomain‐containing protein Eaf3, as a potential mediator of the interaction between the splicing machinery and H3K36 methylation. Here we report our detailed analysis of the mechanism by which this histone modification directly affects spliceosome assembly. Moreover, these studies suggest that subsets of introns may be “tuned” to respond to specific changes in the chromatin environment.

Support or Funding Information

NIGMS grant number GM‐085474

Ruth L. Kirschstein National Research Service Award GM007185
The Role of the Essential Splicing Factor Prp2 in Ribosome Biogenesis
Authors: Edwards Samantha , Hossain Azad , Johnson Tracy
Published: November 2023

Ribosome biogenesis is a highly regulated process. Transcription by RNA polymerases I, II, and III is required to generate the RNA and protein components that comprise the ribosome. In addition, the pre‐ribosomal RNA (pre‐rRNA) transcripts must be processed into the mature 18S, 5.8S, 25S, and 5S rRNAs by multiple exo‐ and endonucleolytic cleavages. Ribosomal protein transcripts are also processed via pre‐mRNA splicing. In the yeast Saccharomyces cerevisiae, pre‐mRNA splicing is integral to the regulation of ribosome biogenesis. In fact, out of ~300 intron‐containing genes in yeast, one third are ribosomal protein genes. As a consequence, the production of ribosomal proteins and functional ribosomes is dependent on the process of pre‐mRNA splicing.

There have been suggestions in the literature that production of rRNA and ribosomal proteins is coordinated to ensure proper production of functional ribosomes. We have recently uncovered evidence that this coordination occurs by tuning rRNA biogenesis with pre‐mRNA splicing. In a genetic screen to identify genes that regulate the activity of an essential splicing factor Prp2, we uncovered numerous suppressors of the high temperature lethality of the prp2‐1 allele. Surprisingly, among these suppressors are mutations at specific sites within the ribosomal DNA locus. Subsequent analysis has revealed cross talk between the machineries that generate and process rRNA and the ribosomal protein transcripts. Our studies suggest that the splicing and rRNA biogenesis machineries are tuned to maintain the proper stoichiometry of these components. Our results also suggest that this stoichiometry is crucial for proper cellular function. We discuss the possible implications of these findings to understanding ribosome biogenesis and human ribosomopathies.

Support or Funding Information

NIGMS grant number GM‐085474

Ruth L. Kirschstein National Research Service Award GM007185
P‐TEFb Regulates Oocyte Maturation and Embryonic Genome Activation by Pol II CTD Phosphorylation and Ribosomal RNA Processing in Mammals
Authors: Jin Dong Il , Oqani Reza K , Lin Tao , Lee Jae Eun , Kim So Yeon
Published: November 2023

Positive transcription elongation factor b (P‐TEFb) is an essential RNA polymerase II carboxyl‐terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. In addition to its role in mRNA production, P‐TEFb is involved in many other cellular events such as DNA damage response and histone modification. Also, recent studies have shown that disruption of P‐TEFb or its homologue in yeast Bur1, impairs ribosomal RNA (rRNA) processing. Here we have investigated the importance of CDK9 kinase subunit of P‐TEFb in early mouse and pig embryo development. We found that CDK9 and its regulatory partner Cyclin T1 are localized into germinal vesicles of oocytes and also embryonic nuclei throughout the preimplantation development. Also, these proteins are co‐localized with nuclear speckles as well as with nucleolar components in oocytes and embryos. Disruption of CDK9 activity by various small molecule inhibitors resulted in defective oocyte maturation and also developmental arrest at the time of embryonic genome activation. Immunolabeling analysis showed that inhibition of CDK9 declines Pol II CTD phosphorylation and nuclear transcription globally in oocyte and embryo. Both nuclear and nucleolar nascent RNAs were negatively affected by CDK9 inhibitors. Also, using RNA fluorescence in situ hybridization for nascent RNA analysis, it was found that inhibition of CDK9 disrupted nucleolar integrity and impairs 28S rRNA production dramatically. Collectively, our data show that P‐TEFb is an important role in both resumption of meiosis and embryonic genome activation, and moreover, CDK9 could be involved in both Pol I‐ and Pol II‐dependent transcription in early mammalian development.
Insights into HuR RRM1–2 tandem domains self‐association and mRNA recognition
Authors: Pinheiro Anderson S. , Lixa Carolina , Jendiroba Kleber A. , Lima Luís Maurício T. R. , de Magalhães Mariana T. Q. , Almeida Fabio C. L.
Published: November 2023

Human antigen R (HuR) employs its mRNA stabilizing activity to function as a major post‐transcriptional regulator of gene expression. This protein plays a central role in the outcome of the inflammatory response as it may stabilize mRNAs of key components of the immune system. HuR comprises three functionally distinct RNA Recognition Motifs: RRM1, RRM2, and RRM3. The two N‐terminal tandem RRM domains can selectively bind AU‐rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly‐A tail of target mRNA. Here, we characterized the structure, dynamics, and binding properties of HuR RRM11‐99 and RRM1–21‐189 tandem domains using a combination of Nuclear Magnetic Resonance and ion mobility‐mass spectrometry. RRM11‐99 adopts a typical RRM fold, composed of two α‐helices and a 4‐stranded β‐sheet (β1α1β2β3α2β4 topology and a β‐hairpin between α2 and β4). 15N relaxation measurements demonstrate that the N‐terminal intrinsically disordered region (residues 1–19) and the RRM1‐RRM2 linker region (residues 95–106) are highly flexible, suggesting that RRM1 and RRM2 domains behave as independent structural modules in solution. NMR titration experiments show that residues on the β‐sheet surface (β1 and β3) and the C‐terminus of RRM11‐99 mediate mRNA binding. An increase in R2/R1 values further confirms the mRNA interaction site, which is compatible with a low μM binding affinity. Both the single RRM11‐99 and the tandem RRM1–21‐189 have the ability to self‐associate, although representing a minor population. Ion mobility‐mass spectrometry identified the presence of oligomers (dimers, trimers and tetramers) with at least three different conformations for the apo forms of RRM11‐99 and RRM1–21‐189. However, only the monomeric species was able to interact with RNA, suggesting that ligand binding shifts the oligomeric/conformational equilibrium of HuR N‐terminal RRM domains. Our results show that the tandem RRM1–2 domains of HuR adopt three distinct conformations in the absence of RNA, suggesting that conformational selection may underlie the mechanism by which HuR recognizes and stabilizes mRNA.

Support or Funding Information

This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by a Brazil Initiative Collaboration Grant from Brown University to A.S.P.
Identification and Characterization of a Minimal Functional Splicing Regulatory Protein, PTBP1
Authors: Ontiveros Robert Jordan , Doan Justin , Adams Eric , Hernandez A L , Black Douglas L , Keppetipola Niroshika M
Published: November 2023

In higher eukaryotes, alternative splicing of a single gene transcript into multiple final spliced mRNA contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that bind to RNA adjacent to regulated exons and influence the assembly of a functional spliceosome at adjacent splice sites. Aberrant alternative splicing has been identified in many diseases such as Alzheimer's disease, muscular dystrophy, and ovarian cancer, underscoring the importance of alternative splicing. Consequently, detailed mechanistic and molecular understanding of how splice variants are generated will provide new targets for therapeutic interventions. The Polypyrimidine Tract Binding Protein 1 (PTBP1) is a well characterized RNA binding protein with roles in alternative splicing regulation, mRNA localization, and IRES‐mediated translation initiation. PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript. PTBP1 is composed of four RNA Recognition Motifs (RRMs) that are joined by three linker peptides. Each RRM can bind to different pyrimidine rich sequences with varying affinity and structural preferences. The complete atomic structure of PTBP1 bound to a target RNA and the interactions between RRM's while bound to RNA is unknown. Attempts to crystallize RNA‐bound PTBP1 have been hindered by the flexible linker regions between RRM1 and 2 (linker 1, 42 aa's) and RRM 2 and 3 (linker 2, 81 aa's). In this study, we aim to identify and characterize a functional minimal‐linker PTBP1 mutant for structure studies via x‐ray crystallography. To this end, a series of PTBP1 mutants were generated using 2‐step PCR with deletions in both linker 1 (Δ19, Δ29, Δ37, Δ40) and 2 regions (Δ21, Δ44, Δ53, Δ68). Mutants were tested for protein expression and splicing repression activity in vivo using mouse Neuro‐2A cells. Western blots indicated that each mutant is well expressed compared to full‐length PTBP1. Splicing assays on three different PTBP1‐regulated test exons reveal that the minimal construct (PTBP1 L1Δ40 ‐ L2Δ68) maintains splicing repression activity comparable to full‐length PTBP1. We have sub cloned PTBP1 L1Δ40 ‐ L2Δ68 into an E. coli expression vector and performed large‐scale protein purification using nickel‐affinity chromatography and dialysis. Purified protein samples indicate that overexpressed PTBP1 L1Δ40 ‐ L2Δ68 is soluble and can be purified in high concentrations for use in x‐ray crystallography. In the future, we intend to further optimize the purification protocol and incorporate size exclusion to increase sample purity.
Thermodynamic Examination of the Bulged‐G Motif in the 23S Ribosomal RNA
Authors: Aman Zachary
Published: November 2023

Ribosomal RNA contains several bulged‐G motifs (also known as sarcin‐ricin loop, loop E or S‐turn) that are formed by six conserved nucleotides, which form an S‐shape in the phosphodiester backbone. The motif provides a characteristic hydration pattern and geometry that is recognized by proteins that bind to it. The 5′ and 3′ nucleotides of the bulged G serve different functions and the two bend angles of the S vary between 20° to 39° (5′ side of the bulged G) and between 11° to 29° (3′ side of bulged G). In the bulged‐G motif derived from the sarcin‐ricin loop of the 23S subunit of the ribosome, the bulged G nucleotide forms a base triple with the U‐A base pair just above it. The base triple is a proposed site for divalent magnesium ion binding. In this study, we are examining the stability of the bulged‐G motif in the presence and absence of magnesium ions. Several modified constructs are also being examined. Thermal denaturation and isothermal titration calorimetry experiments were performed in 1M KCl and 10 mM MgCl2 buffers between pH 5.5 and 7. ΔG°37 of the RNA wild type (WT) construct was −11.2 kcal/mol in potassium ions and increased to −14.0 kcal/mol in 10 mM magnesium ions at pH 7. The WT RNA's interactions with magnesium ions were also studied using isothermal calorimetry and two binding constants, 0.2 and 1 mM were observed in our initial experiments. Modified RNA constructs showed differences in magnesium binding affinity and will be presented. The WT DNA construct showed no differences in stability in 1 M KCl and 10 mM MgCl2.
Cldn18−/− mice reveal novel role for YAP signaling in regulation of distal lung epithelial stem/progenitor cell homeostasis
Authors: Borok Zea , Zhou Beiyun , Flodby Per , Castillo Dan R , Luo Jiao , Yu Fa‐Xing , McConnell Alicia , Varghese Bino A , Li Guanglei , Conti Peter S , Liu Yixin , Sunohara Mitsuhiro , Liebler Janice M , Yang Chenchen , Marconett Crystal N , Laird‐Offringa Ite A , Minoo Parviz , Guan Kun‐Liang , Stripp Barry R , Crandall Edward D
Published: November 2023

Claudins are integral tight junction (TJ) proteins that contribute to cell polarity and regulate paracellular permeability to ions and solutes. Claudin 18 (CLDN18) is one of the most highly expressed claudin family members in lung alveolar epithelium. To investigate the role of CLDN18 in alveolar homeostasis, we recently generated Cldn18−/− mice that demonstrate increased lung solute permeability and alveolar fluid clearance (AFC) compared to wild type (WT) controls. Lungs of Cldn18−/− mice are markedly enlarged due to increased abundance and proliferation of alveolar epithelial type II (AT2) cells, known progenitors of distal lung epithelium, with resultant parenchymal expansion. Cldn18−/− AT2 cells grown with MLg fibroblasts in 3‐dimensional (3D) culture show increased colony forming efficiency (CFE), suggesting increased progenitor capacity. Given its known role in regulating stem/progenitor cell proliferation and organ size, we investigated a potential role for Yes‐associated protein (YAP) signaling in mediating the proliferative phenotype of Cldn18−/− lung progenitors. Progenitor cell activation was accompanied by activation of YAP, as evidenced by increased nuclear YAP, increased expression of YAP target genes in lungs of Cldn18−/− mice, and increased YAP and decreased phospho‐YAP (p‐YAP) by western analysis in isolated Cldn18−/− AT2 cells and by immunofluorescence in Cldn18−/− AT2 cells in 3D culture. Treatment with the YAP inhibitor verteporfin (VP, 100 mg/kg) reduced AT2 cell proliferation in vivo (EdU+NKX2.1+/total cells: 1.36 ± 0.10% vehicle vs 0.93 ± 0.06% VP, p<0.05) and decreased lung size (lung dry weight/body weight ratios (mg/g): 3.56 ± 0.08 vehicle vs 2.93 ± 0.13 VP, p<0.05) in Cldn18−/− mice. Inhibition of YAP with VP (0.75 μM) or shRNA decreased colony size (~50% vs control) and number (30–50% vs control) and reduced proliferation (Ki67+ cells at 10.7 ± 0.4% VP vs 29.2 ± 3.7% vehicle) of Cldn18−/− AT2 cells in 3D culture, while overexpression of CLDN18 decreased YAP nuclear localization, cell proliferation, CFE and YAP activity. These results reveal a novel role for YAP signaling in regulation of distal lung epithelial progenitor cell homeostasis and identify a role for TJ proteins, in particular CLDN18, in regulating YAP activity and organ size. Overall, they suggest a mechanism whereby growth‐promoting signals are transduced from TJ to the nucleus that has important implications for modulating stem/progenitor cell function and regeneration following injury.

Support or Funding Information

National Institutes of Health, Hastings and Whittier Foundations
YAP1 stimulates angiogenesis and lung regeneration through the Angiopoietin‐Tie2 pathway
Authors: Mammoto Akiko , Mammoto Tadanori
Published: November 2023

Angiogenesis, the formation of new blood capillaries, plays key roles in organ development and regeneration. Inhibition of angiogenesis through the blockade of angiogenic signaling pathways impairs adult lung regeneration. Previously we have reported that Wnt co‐receptor, low‐density lipoprotein receptor‐related protein 5 controls lung vascular and alveolar regeneration. The Hippo signaling transducer, Yes‐associated protein (YAP1), cooperates with Wnt‐b‐catenin signaling and controls organ size and regeneration. Here, we have demonstrated that YAP1 knockdown decreases angiogenic factor receptor Tie2 expression and inhibits endothelial cell sprouting and lung epithelial morphogenesis in vitro. YAP1 knockdown also attenuates angiogenesis and alveolar epithelial cell recruitment into the hydrogels implanted on the mouse lungs and inhibits compensatory lung growth after unilateral pneumonectomy in adult mice. These findings suggest that YAP1 plays an important role in lung vascular and alveolar regeneration and modulation of YAP1 expression in alveolar capillary endothelial cells may be a novel strategy for lung regeneration.

Support or Funding Information

Boston Children's Hospital Career Development Awards
Messenger RNA Stability Drives the Expression of Antibiotic Resistance Methyltransferase Independently of Ribosome Stalling
Authors: Los Katia , Yap M.‐N. Frances
Published: November 2023

Macrolides, lincosamides, and streptogramin B (MLSB) are structurally distinct antibiotics that are clinically successful in treatment of bacterial infections. Their efficacy has decreased due to the rapid emergence of resistant bacterial strains. Cross‐resistance to MLSB antibiotics often arises through the production of erythromycin resistance N‐6‐methyltransferase (Erm). The enzyme modifies the common MLSB binding site within the ribosomal exit tunnel and reduces the drug binding affinity to the ribosome. Previous model based on the ermCL‐ermC system suggests that the synthesis of Erm is induced through the antibiotic‐stalled ribosome that translates the co‐transcribed upstream leader sequence. Ribosome stalling induces a structural rearrangement of erm transcript and exposes the otherwise masked erm Shine‐Dalgarno (SD) sequence, subsequently leading to translational initiation. Curiously, many resistant clinical isolates carry mutated versions of leader sequence, including the ribosome‐stalling null mutants. The seemingly paradoxical role of leader peptide‐dependent ribosome stalling in MLSB resistance remains poorly understood.

We used the ermBLEF‐ermB operon from the human pathogen Staphylococcus aureus as a model to determine how the expression of ermB, unlike other homologous erm genes, is inducible by a broader spectrum of MLSB antibiotics. Our data show that disrupting the translation of ErmBL abolishes ribosome stalling but results in high basal levels of ErmB that is well correlated with elevated ribosome methylation and antibiotic resistance, suggesting that a ribosome stalling‐independent determinant is required for ermB expression. Subsequently, we found that both MLSB antibiotic treatment and abrogating ErmBL translation upregulate the basal ermB mRNA levels and prolong the ermB mRNA half‐life. Furthermore, antibiotic exposure reduces mRNA cleavage at a site immediately downstream of the ermB SD sequence. The data confirmed that the expression of ermB could be upregulated by antibiotics via stabilization and accumulation of its mRNA. Finally, genome‐wide transcriptome analysis showed that the stabilizing effect of antibiotics on mRNA is not limited to ermBL‐ermB; antibiotics representing a ribosome stalling inducer and a non‐inducer co‐regulate similar sets of genes and increase the half‐lives of the majority of analyzed transcripts.

Collectively, our data suggest a model by which the upregulation of ErmB is achieved through disrupted translation of the leader peptide, which normally acts as a break to fine‐tune the cellular ErmB levels. Tight regulation of ErmB is necessary because hypermethylation of ribosome may impose a fitness cost on the bacterium.

Support or Funding Information

This work is supported by the NIH, the Pew Charitable Trust (Pew Scholars in the Biomedical Sciences), the Edward Mallinckrodt Jr. Foundation, and SLU President's Research Fund.
Regulation of Protein Synthesis by Post‐translational Modification of Eukaryotic Translation Elongation Factor 1A
Authors: Sharma Pragati , Mateyak Maria K , He Donming , Perez Winder B , Kinzy Terri G
Published: November 2023

Protein synthesis is an intricate multi‐step process accomplished through a series of concerted actions of several different factors. Post initiation, translation elongation continues in a cyclic manner with the delivery of cognate aa‐tRNA to ribosomal A site, peptide bond formation and translocation to P‐site. The broad mechanism of action of elongation factors is known, however the regulation of translation elongation remains poorly understood. To safeguard timely and accurate gene expression, the protein synthesis machinery must be strictly regulated. Indeed the eukaryotic Elongation Factor 1A (eEF1A) shows extensive post‐translational modifications, both phosphorylation and methylation. In Saccharomyces cerevisiae, global proteomic analyses have reported at least 15 residues in eEF1A that are phosphorylated under different growth conditions. We have created a set of corresponding alanine (non‐phosphorylatable) and aspartate (phosphomimetic) mutations in these residues to study their phenotypic as well as enzymatic effects. Our in vivo and in vitro analyses in budding yeast show that S53D, Y416D and T430D mutant alleles are unable to support cell growth as the only form of eEF1A, while S18D, T259D and Y416A show slow growth and an initiation defect manifested by Gcn2 activation. Further experiments to test the translation fidelity, in vitro enzymatic activity and their ability to interact with cofactors are ongoing. In addition to translation elongation, eEF1A has been implicated in a wide variety of cellular processes, for example cytoskeleton organization through actin bundling, protein quality control, viral invasion and replication, etc. This work is aimed at identifying the events and factors that modulate eEF1A activity ensuring careful control of gene expression and appropriate cell function.

Support or Funding Information

NIH grant RO1 GM057483