bluuebunny/crossref_metadata_2025_split huggingface dataset:
Retention of CENP‐A nucleosomes during DNA replication
Authors: Zasadzinska Ewelina , Huang Jiehuan , Bailey Aaron O. , Lee Nancy S. , Wong Kelvin A. , Jakubaszek Marta R. , Guo Lucie , Black Ben E. , Foltz Daniel R. 
Published: November 2023

Centromeres are chromosomal loci that define the site of kinetochore formation and ensure equal chromosome segregation during mitosis and genomic stability. Centromeric identity is epigenetically regulated by the incorporation of the nucleosome containing the centromere specific CENP‐A histone H3 variant. In contrast to the canonical H3.1, CENP‐A deposition is independent of DNA replication, and occurs in early G1. Therefore new CENP‐A deposition and retention of CENP‐A during S‐phase are key events in epigenetic inheritance. DNA replication presents a challenge for CENP‐A inheritance because nucleosomes are removed to allow passage of the replication fork. However, CENP‐A nucleosomes are stably retained across S‐phase. We hypothesize that CENP‐A retention at centromeres during S phase is facilitated by a unique mechanism that distinguishes CENP‐A containing nucleosomes from bulk histones. We fused CENP‐A to the promiscuous biotin ligase (BirA*) to chemically label and affinity purify proteins transiently associated with CENP‐A during S‐phase. SILAC labeling and Mass Spec were used to identify S‐phase specific interactions. This analysis identified known G1 coupled CENP‐A deposition proteins such as HJURP and Mis18BP1 as well as novel factors, as associating with CENP‐A during S‐phase. Therefore the CENP‐A deposition proteins appear to play dual roles in S‐phase and G1. We demonstrate that HJURP is required for the inheritance of existing CENP‐A nucleosomes during DNA replication. MCM2 subunit of the replicative helicase binds HJURP and directly interacts with CENP‐A. Our data suggests that mechanism of transmission of CENP‐A nucleosomes requires CENP‐A specific deposition machinery including HJURP together with the activity of MCM2.Support or Funding InformationAHA Predoctoral Fellowship
Sequence Analysis of Mutant Chick Embryo DNA
Authors: Vo Jessica , Byk Jennifer , Smith Sheila R. , Benore Marilee Ann 
Published: November 2023

Riboflavin Binding Protein is a transport glyco‐phospho‐protein found in all oviparous species, required for transport of the vitamin riboflavin needed for proper embryonic development. A mutant strain of chickens cannot synthesize the protein; the embryos lacking riboflavin die after the thirteenth day of incubation. DNA was extracted from the embryos. Several kits were utilized and compared to determine the best method to isolate pure DNA in large yield. Sequence comparisons to wild‐type white leghorn chickens are used to confirm the DNA sequence causing the mutation.Support or Funding InformationUniversity of Michigan Dearborn Campus Grants
DNMTi Decitabine Induces a Type I Interferon Response in Leukemia Cell Lines
Authors: Nicholas David J , Srivastava Pragya , AlShangity AlMujtaba , Nemeth Michael J 
Published: November 2023

Decitabine (DAC) is an FDA approved DNA methyltransferase inhibitor (DNMTi) currently used for treatment of Myelodysplastic Syndrome and Acute Myeloid Leukemia. Only 40% of patients respond to DAC treatment. Therefore, discovering and enhancing DAC's mechanism of action is of paramount importance. Recent literature suggests that DNMTis demethylate and induce expression of latent dsRNA retroviral species in ovarian cancer and colorectal cell lines. This viral mimicry triggers an interferon response that results in both apoptosis and targeting of treated cancer cells. To test whether a similar mechanism of action occurs in myeloid cancer, the leukemia cell lines U937, HL60, and KG1a were evaluated for gene expression changes in the interferon response pathway after two treatments of 0.5um DAC using RTqPCR. Up regulation of Interferon response genes DDX58, MDA5, OASL, MAVS, ISG15, IFN B, and IRF7 were observed in KG1a cells with reported fold changes of 3.45, 1.88, 54.40, 2.04, 3.25, 5.88, and 80.00 respectively. In addition, the ERV MLT1C49 was upregulated by a fold change of 2.46. In the U937 cell line, DDX58, MDA5, OASL, MAVS, and ISG15, were upregulated by 1.59, 2.02, 3.62, 1.468, and 6.3646, respectively. For the HL60 cell line, DDX58, OASL, and ISG15 were upregulated by 1.79, 5.16, and 1.87, respectively. In summary, our data suggests Decitabine induces a type I interferon response in leukemia cells, giving promise to the combinatorial treatment of DAC with emerging immunotherapy treatments.Support or Funding InformationNIH R25CA181003 and SUNY Geneseo Biology Department
Epigenetic Regulation through UHRF Proteins
Authors: Rothbart Scott B , Vaughan Robert M , Cornett Evan M , Dickson Bradley M 
Published: November 2023

The epigenetic inheritance of DNA methylation requires UHRF1, a histone‐ and DNA‐binding RING E3 ubiquitin ligase whose misregulation may contribute to the hallmark aberrance of DNA methylation patterning seen in cancer. UHRF1 facilitates recruitment of DNMT1 to replicating chromatin through orchestrated recognition of histones and DNA, which converge on ubiquitination of histone H3, a binding site for DNMT1. UHRF2 shares a high degree of structural homology with UHRF1, but surprisingly lacks functional redundancy as a DNA methylation regulator. Through comprehensive and comparative biochemical analysis of UHRF1 and UHRF2, we uncover mechanisms of intra‐ and inter‐molecular crosstalk regulating the binding and enzymatic activities of these chromatin regulators and reveal a molecular mechanism that uncouples UHRF2 from the DNA methylation maintenance program.Support or Funding InformationThis work was supported in part by the Van Andel Research Institute and a research grant from the National Institutes of Health to S.B.R. (Grant No. CA181343).
Unraveling The Role of Helicases in Nonfunctional rRNA Decay
Authors: Yu Victor , LaRiviere Frederick 
Published: November 2023

rRNAs are essential, long‐lived, and highly abundant, comprising up to 90% of total cellular RNA. However, when structurally intact rRNAs in Saccharomyces cerevisiae are functionally compromised, they are instead rapidly degraded through a quality control pathway termed nonfunctional ribosomal RNA decay (NRD). Currently, two mechanistically distinct types of NRD are known: 18S NRD and 25S NRD. 18S NRD is translation dependent and removes rRNAs with loss of function mutations in the small subunit decoding site. The 18S NRD mechanism involves the translation factor‐like protein Dom34 and its binding partner Hbs1. On the other hand, 25S NRD, which occurs prior to subunit joining, is translation independent and removes nonfunctional rRNAs containing mutations in the large subunit peptidyl transferase center. The 25S NRD mechanism involves polyubiquination of proteins associated with a nonfunctional 25S rRNA through the E3 ligase components Rtt101 and Mms1. While some of the proteins that are involved in either 18S or 25S NRD are known, we have not yet elucidated the complete molecular mechanism of NRD. Since RNA helicases have been implicated in other RNA degradation pathways, we hypothesize that one or more helicases may be important for either or both NRD mechanisms. S. cerevisiae contains 44 different helicases, 14 of which are non‐essential. Thus, we are initially investigating whether any of these 14 helicases are involved in NRD using single gene deletion yeast strains. We transformed BY4741 and the 14 non‐essential helicase knock‐out strains with various rRNA expression plasmids that encode for functional and/or nonfunctional versions of the 18S and 25S rRNAs. These plasmid‐derived rRNAs also contain short, unique sequence tags that allows for specific detection of these rRNAs in the presence of total cellular rRNA. We measured the growth phenotypes of these transformed strains at various temperatures in liquid cultures. At standard temperatures, we see a slow growth phenotype in some of the deletion strains transformed with either nonfunctional 18S or 25S rRNA expression plasmids. Since some helicase deletion strains exhibit either cold‐ or temperature‐sensitivities, we are currently measuring growth phenotypes at these normally restrictive temperatures. In addition, we are measuring the rRNA expression levels in these deletion strains using an RT‐qPCR assay to quantify the level of NRD activity. We hope this work will provide insight into the role of helicases in RNA degradation mechanisms and contribute to our understanding of NRD.Support or Funding InformationThis work is supported by the Howard Hughes Medical Institute.
Biochemical Characterization of Evolutionary Divergent Vertebrate LARP6 Proteins
Authors: Castro Jose Miguel , Horn Daniel A , Lewis Karen A 
Published: November 2023

The La‐related proteins (LARPs) contain a conserved bipartite RNA binding domain, called the La module, which consists of the La motif and an RNA recognition motif (RRM). Across the different LARP subfamilies, the La module interacts with a variety of RNAs, allowing this protein superfamily to perform roles as chaperone proteins, post‐transcriptional regulatory factors, regulators of RNA metabolism, and perhaps even tumor suppressors. The ligand specificity of the LARP6 subfamily is still unknown. The only endogenous ligand for LARP6 is found in the 5′ untranslated region of collagen type I mRNA. Animal studies confirmed that the binding of a particular RNA structure by LARP6 was critical for collagen production and muscle development. In this study, we are using the LARP6 proteins from zebrafish (Danio rerio), platyfish (Xiphophorus maculatus) and human to carry out detailed biochemical analyses of vertebrate LARP6 RNA binding activity. The purpose is to determine if there is a species‐specific protein‐RNA interaction due to co‐evolution of the ligand‐binding surface within the LARP6 RRM and the RNA ligand sequence and/or structure. Recombinant fish and human LARP6 proteins were expressed in E. coli with an N‐terminal SUMO tag. A filtration screening assay was used to identify additives that increase solubility and stability to the fish proteins. In order to robustly compare the binding affinity of the fish proteins to the human LARP6, we then optimized conditions for SUMO cleavage by Ulp1. Limited proteolysis studies were used to compare the domain topology of the fish LARP6 proteins to the human homolog. The RNA binding activity of all LARP6 proteins for the COL1A1 stem loop RNA from the appropriate species was measured by electrophoretic mobility shift assays (EMSAs). In order to determine if the La motif and RRM co‐evolved within species, interspecies chimeras were produced. These studies will explore whether the RRM determines the ligand specificity of the LARP6 protein.Support or Funding InformationFunding provided by the Texas State University Department of Chemistry and Biochemistry; the National Institutes of Health through the South Texas Doctoral Bridge Program (GM102783 to J.M.C.) and an AREA grant (GM119096 to K.A.L.).
Understanding the role of post‐translational modifications on the splicing activity of two related RNA binding proteins
Authors: Reynaga Janice Mireya , Pina Jeffrey , Keppetipola Niroshika 
Published: November 2023

The process of alternative splicing is regulated in part by RNA Binding Proteins, many of which occur as gene families with members sharing high primary structure identity and similar domain organization but have tissue specific expression patterns and different splicing activities. The Polypyrimidine Tract Binding Protein 1 (PTBP1) and Polypyrimidine Tract Binding Protein 2 (PTBP2) are paralogous RNA binding proteins. The two proteins share 74% primary structure identity and similar domain organization with 4 RNA binding domains connected via 3 linker regions. Albeit the structural similarity, PTBP1 and PTBP2 show differences in their splicing repression activity, where some exons are repressed by both and others repressed more strongly by PTBP1. The two proteins also have non‐overlapping expression patterns where PTBP1 is expressed in neuronal progenitor cells but is replaced by PTBP2 in differentiating neurons. This change in protein expression levels is accompanied by a transition in the neuronal splicing program that is critical for neuronal development and maturation. Previous studies investigating regions of primary structure that dictate higher PTBP1 repression activity on certain exons such as the c‐Src N1 exon highlighted that multiple regions confer splicing repression on PTBP2. Recent reports indicate that the two proteins are post‐translationally modified. Thus, we hypothesize that post‐translational modifications dictate the different splicing activity of PTBP1 and PTBP2. To test this, we have overexpressed PTBP1 and PTBP2 in mouse neuroblastoma (neuro 2A) cells and probed for protein phosphorylation, via Phos‐Tag Gel Electrophoresis and for ubiquitination and acetylation via Western Blot. In parallel, we purified Flag‐tagged PTBP1 and PTBP2 via Flag‐Immunoprecipitation and analyzed the proteins for PTM's via Mass Spectrometry. Preliminary results indicate that the two proteins are modified in neuro 2A cells. We are currently conducting site‐directed mutagenesis of modified residues to understand the role of these modifications on PTBP1 and PTBP2 splicing activity.Support or Funding InformationThis work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐20]
Thermodynamic analysis of a 4×4 internal loop in magnesium riboswitch
Authors: Gilbertson Ellen 
Published: November 2023

A riboswitch is a segment of noncoding RNA that regulates the transcription of a gene in response to external cellular events. The magnesium riboswitch specifically regulates the expression of a transmembrane magnesium transport protein, mgtA, in response to intracellular magnesium ion concentration. The magnesium riboswitch in Salmonella enterica is a 161‐nucleotide sequence that exhibits conformational change upon magnesium binding. A 2.6 Å resolution crystal structure shows 6 magnesium‐RNA binding sites. This study used thermal denaturation and isothermal titration calorimetry to characterize the thermodynamic properties of Core 2, where two of the six magnesium ions are expected to bind. The Core 2 wildtype RNA construct conserves the adenine‐rich 4×4 internal loop sequence, including the magnesium‐binding sites A63 and A80. Modifications to positions 63 and 80 were also designed. All constructs were thermally denatured in 1 M KCl and 10 mM MgCl2 buffers at pH 5.5 and 7.0. In 1 M KCl, the RNA wildtype construct is more stable at pH 5.5 than at pH 7.0 by 1.5 kcal/mol, indicating a potential protonation‐dependent hydrogen bond. The wildtype gained ~3 kcal/mol of stability in 10mM magnesium ions as compared to 1M KCl in both pH buffers, as might be expected for a magnesium riboswitch. The magnitude of these interactions provides a benchmark for understanding the contributions of magnesium ions to RNA stability. Modifications of adenine at positions 63 and 80 also showed greater stability at the lower pH. These modified constructs showed a significant loss of the additional stabilization in magnesium over 1 M KCl at both pH conditions. Isothermal analyses of magnesium binding to RNA are also being performed. Thermodynamic examination of wild type and modified RNA constructs and their stability in magnesium ions will be presented.Support or Funding InformationGrover Laboratory at Colorado College, Colorado College Department of Biochemistry, Colorado College Venture Grant, Crecelius Family Award for Outstanding Research in Biochemistry
Enhancement of Transgene Production in a Vaccinia Virus‐based Expression System
Authors: Richard Brent , Flores Julia , Jacobs Bertram , Chaput John , Wellensiek Brian 
Published: November 2023

Poxvirus‐based vaccinations, i.e., vaccinia viruses (VACV), have been used more extensively for human immunization than any other vaccine in human history. VACV‐based vaccines are of particular interest due to the relative ease with which the VACV genome can be modified to express genes from a variety of sources. In recent years, there have been great advances in VACV‐based vaccines, including the generation of replication‐competent (non‐attenuated) and replication‐incompetent (attenuated) strains of VACV. While non‐attenuated VACV are highly immunogenic due to their ability to replicate in human cells, vaccines generated using these strains have higher than normal complication rates and are restricted in the population that can receive them. Attenuated strains lack the ability to replicate in human cells, resulting in a dramatic decrease in side effects, however this increase in safety comes at the cost of a reduced immune response. To address this issue, our current research is focused on increasing viral protein production in an attenuated VACV. In previous studies, we have discovered a human translation enhancing element (hTEE‐658) capable of serving as a VACV promoter. Characterization of this sequence has demonstrated that when this element is used in a VACV‐based expression system, it leads to increased protein expression levels over those driven by standard VACV promoters. Further optimization of expression levels driven by hTEE‐658, through varying the length of the transgene leader sequence, has resulted in the generation of a sequence element capable of driving high levels of protein production in an attenuated VACV system. These results provide a promising option for the production of safe, highly immunogenic VACVs that can be used to develop vaccines against a wide range of infectious diseases.Support or Funding InformationFunding for this research was provided by Midwestern University intramural funds awarded to BPW and an NIH grant awarded to JCC
Phosphorylation of eIF2 directs multiple mechanisms of preferential translation for cell adaptation to environmental stress
Authors: Wek Ronald C , Young Sara K , Willy Jeffrey A , Fusakio Michael E 
Published: November 2023

Phosphorylation of eIF2 (eIF2‐P) lowers protein synthesis to conserve resources and facilitate preferential translation of specific transcripts that promote adaptation to environmental stresses. Central to the mechanisms of preferential translation are upstream ORFs (uORFs) embedded within the 5′‐leaders of mRNAs, which serve as “bar codes” that help ribosomes determine the levels of translation of mRNA coding sequences (CDS) during eIF2‐P. We have been studying the functions of uORFs that demarcate translational control and the roles that these preferentially translated genes play in cell adaptation to stress. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) each function to dephosphorylate eIF2‐P and restore protein synthesis. Both GADD34 and CReP mRNAs contain two upstream uORFs, and using biochemical and genetic approaches we show that distinct features of these uORFs are critical for differential CDS translation. In the absence of stress and low eIF2‐P, translation of an inhibitory uORF in GADD34 acts as a barrier by inefficient translation termination that prevents subsequent reinitiation at the GADD34 CDS. Induced eIF2‐P during stress directs ribosome bypass of the inhibitory uORF, leading to enhanced translation of the GADD34 CDS. By comparison, CReP expression occurs independent of eIF2‐P via an uORF that allows for translation reinitiation at the CReP CDS independent of stress conditions. Alterations in the GADD34 uORF not only affect the status of eIF2‐P and translational control, but also cell adaptation to stress. Translation of other preferentially translated genes also feature bypass of uORFs, although by mechanisms featuring different uORF properties. For example, bypass of a single uORF directs preferential translation of CHOP (DDIT3/GADD153), a transcription factor that triggers cell death during periods of extended stress. We discovered that the CHOP uORF serves to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP CDS. Deletion of the CHOP uORF increases the levels and alters the pattern of induced CHOP expression during eIF2‐P. Elevated CHOP expression leads to increased cell death in response to stress. Finally, we determined that bypass of uORFs in IBTKalpha enhances translation of the CDS in response to eIF2‐P. Elevated levels of IBTKalpha localize to endoplasmic reticulum exit sites and facilitate initiation of autophagy. These examples and others indicate that the proper mixing and matching of uORFs with distinct properties are central for directing gene‐specific translation during eIF2‐P. Preferentially translated genes are critical for the timing and extent of translation and transcription, and alter metabolism and reclamation processes such as autophagy, which together can determine cell survival during environmental stresses.Support or Funding InformationNIH GM049164
A high‐throughput approach to annotate the lysine methylome
Authors: Cornett Evan M , Dickson Bradley M , Krajewski Krzysztof , Cowles Martis W , Sun Zu‐Wen , Rothbart Scott B. 
Published: November 2023

Lysine methylation is a widespread posttranslational modification (PTM) in eukaryotes whose protein regulatory functions have been illuminated through studies of histone lysine methylation and its key connections to chromatin and epigenetic regulation. Beyond histones, a major gap in knowledge is broadening between the rapidly expanding lysine methylome and the proteins responsible for the addition, removal, and recognition of these PTMs. As several inhibitors of lysine methylation regulators are in preclinical and clinical development for cancer therapy, defining the full target spectrum of these proteins is critical for understanding mechanisms of action for these drugs, to rationalize potential combination therapies, and to identify reliable biomarkers for clinical activity. Here we debut the development of a high‐throughput and unbiased functional proteomics platform that queries the activities of lysine methyltransferases (KMTs) and generates molecular footprints that can be mapped to protein targets. We demonstrate the utility of this platform towards the discovery of novel substrates for KMTs. Full implementation of this platform will provide critical systems level information to enable the construction of lysine methylation signaling networks and will facilitate fundamental understanding of lysine methylation function in human health and disease.Support or Funding InformationVan Andel Research Institiute, Van Andel Institute Employee Impact Campaign, and EpiCypher