bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rabbit Polyclonal Antibodies to Detect C7orf49 Proteins Toward Understanding Double Strand Break Repair
Authors: Englund Bailey 
Published: November 2023

DNA double‐strand breaks are a highly cytotoxic form of DNA damage. Non‐homologous end joining (NHEJ) is an important mechanism that repairs double‐strand breaks in DNA. Accurate function of NHEJ protects genome integrity; however, malfunctions within the mechanism can lead to genomic changes resulting in carcinogenesis. The basic requirements for the NHEJ process have been determined, but there are still questions about the step‐wise assembly of the system. Our preliminary data indicates chromosome 7 open reading frame 49 (C7orf49) produces short open reading frame (sORF)‐encoded proteins that may participate in non‐homologous end joining (NHEJ). C7orf49 encodes three alternatively spliced protein isoforms, which will be referred to as spliced forms 1, 2 and 3 (SF‐1, SF‐2 and SF‐3, respectively). Transcriptome analysis and ribosomal profiling indicate that the C7orf49 locus is both transcribed and translated in human cells; but it is presently unclear: 1) whether endogenously expressed C7orf49‐encoded proteins are stable or degraded, 2) how abundant C7orf49‐encoded proteins are, 3) what isoforms are expressed, and 4) how DNA damage affects the abundance of C7orf49‐encoded proteins. The goal of this project is to determine whether our anti‐C7orf49 antibodies, rabbit polyclonal antibodies, can be used to detect C7orf49‐encoded proteins in human cell lysates. Antibodies were tested via Western blot under variable conditions with both wild type and C7orf49‐deficient HEK293 cells. C7orf49 antisera recognized SF‐1 and SF‐2 as recombinant purified proteins or when ectopically expressed in HEK293 cells. Compared to pre‐immune sera, immune sera recognized bands in wild type cell extracts, which may be encoded by C7orf49. Future experiments will investigate which isoforms are expressed, and what affects DSB‐inducing drugs have on C7orf49‐encoded protein abundance, to help determine how these proteins contribute to NHEJ.Support or Funding InformationSupport received from Leslyn Hanakahi and Matthew Summerlin at University of Illinois Chicago ‐ Rockford
DNA Repeat Expansion and Mismatch Repair: A Recurrent Problem
Authors: Fuselier Kayla T B , Grabczyk Ed 
Published: November 2023

At least 22 human neurodegenerative diseases are caused by DNA repeat expansion. Repeat expansion can occur at any developmental stage or during the life of an affected individual. Somatic repeat expansion is particularly interesting because it has the potential to affect disease age‐of‐onset, severity, and progression. Thus, understanding the factors that cause this somatic expansion is crucial and makes it a prime therapeutic target for these disorders. Previous studies have found that a major driver for repeat expansion is DNA mismatch repair (MMR). Though MMR is important for genomic stability, it has been conversely associated with trinucleotide repeat instability.Our lab has focused on the GAA·TTC trinucleotide repeats that are associated with Friedreich ataxia (FRDA), a progressive neurodegenerative disorder that currently has no treatment. Our objective is to refine our understanding of the role MMR proteins play in the repeat expansion that causes FRDA. We have previously shown that the expansion rate is associated with transcription within the repeat and requires the MMR recognition complex MutSβ and the subsequent action of a MutL endonuclease complex. In our human cell model, we have established that the necessary endonuclease complex is MutLγ, a heterodimer of MLH1 and MLH3. Our research indicates a pivotal role for MLH3 in GAA·TTC expansion, specifically a single isoform, MLH3 isoform 1 (MLH3 iso1). Humans have two splice variants for MLH3; MLH3iso1 includes exon 7, an endonucleolytic domain, and MLH3iso2 that excludes exon 7. All known MutL complexes require MLH1, making it a poor therapeutic target. In contrast, MLH3 is a constituent of only MutLγ, a minor form expressed as a small percentage of total MutL complexes. Therefore, MLH3 isoforms are a much more attractive target to halt GAA·TTC expansion rather than pursuing other DNA mismatch repair enzymes.Support or Funding InformationFriedreich Ataxia Research Alliance (FARA)
Activation of Transcription‐Coupled 5′‐RNA Capping by TFIIH
Authors: Noe‐Gonzalez Melvin , Conaway Joan , Conaway Ronald 
Published: November 2023

Transcription is coupled in time and space to various mRNA processing events including 5′ capping, splicing, and polyadenylation. Capping of the 5′ end of pre‐mRNA, the first detectable mRNA processing event, is thought to occur by the time the transcript is only 25–30 nucleotides long and correlates with hyperphosphorylation of the C‐terminal domain (CTD) of transcribing Pol II. Notably, previous studies have shown that capping is over 100‐fold more efficient when coupled to Pol II transcription.Here we exploit a highly purified, reconstituted Pol II transcription system to investigate the mechanism of co‐transcriptional capping. We present direct evidence that TFIIH functions as a potent activator of co‐transcriptional capping in this system. The TFIIH‐associated CAK protein kinase most likely contributes to TFIIH action in transcription‐coupled capping in light of evidence (i) that CAK inhibitors, H8 and THZ1, inhibit TFIIH function and (ii) that recombinant CAK can at least partially replace TFIIH. Finally, we use DNA:RNA scaffolds to show that capping of transcripts in artificial Pol II elongation complexes also requires TFIIH and its CAK kinase in the absence of other general transcription factors.Taken together, these data support the model that TFIIH‐dependent phosphorylation, most likely of the Pol II CTD, plays an integral role in transcription‐coupled RNA capping.
The Heme‐Binding PAS Domain Mediates Dimerization in the CO‐Sensing Transcription Factor BxRcoM‐1
Authors: Pinhancos Rebeca C. , Bowman Hannah E. , Dent Matthew R. , Young Byron H. , Berndsen Christopher E. , Burstyn Judith N 
Published: November 2023

The gases NO, O2, CO and H2S are small‐molecule effectors involved in signaling pathways. Heme serves as an effector‐binding site in many proteins that sense these molecules, including gas‐sensing transcription factors, and the effector‐binding signal is transduced through varying allosteric mechanisms. Bacterial transcription factors are excellent systems in which to study gas‐mediated allostery due to their simplicity relative to the multi‐protein complexes involved in eukaryotic transcription. RcoM‐1 is a heme‐containing, CO‐responsive transcription factor that appears to regulate aerobic CO oxidation in the non‐pathogenic bacterium Burkholderia xenovorans. The RcoM‐1 heme plays a key role in CO‐sensing. Under reducing conditions, the Fe(III) heme undergoes a redox‐mediated ligand switch in which a neutral donor replaces an axial cysteine(thiolate) ligand, Cys94. The neutral donor ligand Met104 was identified through a spectroscopic study of three Met‐to‐Leu variants. RcoM‐1 variants in which Met104 is replaced with a non‐coordinating Leu residue exhibited electronic absorption and magnetic circular dichroism spectra that, under reducing conditions, are indicative of a five‐coordinate, high‐spin (S = 2) Fe(II) heme center. CO replaces Met104, binds to the Fe(II) heme, and presumably modulates DNA‐binding activity. The structure of RcoM‐1 is still unknown; however, sequence homology suggests that RcoM‐1 is a unique, single‐component signaling protein that contains both a PAS‐like heme‐binding domain and a LytTR‐like DNA‐binding domain. We probed the oligomeric status and topology of this novel single‐component transcription factor. Size exclusion chromatography (SEC), SAXS, and analytical ultracentrifugation data (MW/MS = 2.25 ± 0.01) are consistent with a dimeric structure for RcoM‐1. Further SEC data show that an RcoM‐1 truncate containing only the heme domain is dimeric and that variants lacking the His74 ligand, which bear no heme, are also dimeric. Taken together, these data suggest that RcoM‐1 dimerizes via its heme domain, but dimerization does not require heme. A stable, dimeric RcoM‐1 homology model that dimerizes via the heme domain was built that fits well within the molecular envelope predicted by SAXS.Support or Funding InformationThe National Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)
Transcriptome Analysis of Conditional Knockout Mice
Authors: Harvey Amanda L , Biefeld Chelsea , Sztroin Kelcie , Zhu Julia , Yu Gang , Dries Daniel R. 
Published: November 2023

Gamma‐secretase is an intramembrane protein that cleaves type I transmembrane proteins. It is known to be involved in Alzheimer's disease and neurodevelopment. Previous studies have investigated the role of gamma‐secretase role in neurons. However, it has seldom been considered in glial cells, the non‐neuronal cells of the brain. Here, a conditional knockout mouse was made in which Nicastrin, a substrate receptor for gamma‐secretase, was rendered nonfunctional in oligodendrocytes. These mice displayed schizophrenia‐ and compulsive‐like phenotypes. In order to identify genes and pathways that elicit these phenotypes, RNA‐Seq was used to perform transcriptome analysis searching for differentially expressed genes in the experimental mice. Gene ontology programs were used to evaluate the genes and identify biological processes that may play a role in the behavioral aspects of these mice. Disease pathways identified in the analysis include Alzheimer's disease, myelination, schizophrenia, and oligodendrocyte function. Cellular pathways include extracellular communication and immune responses. Together this analysis presents a picture of the cellular pathways involved in the non‐cognitive aspects of Alzheimer's disease as it relates to the non‐neuronal white matter of the brain.Support or Funding InformationAcknowledgments We would like to thank the Howard Hughes Medical Institute, the National Institutes of Health (Grant R01 AG029547), and the International Healthspan Institute for providing funding.
Using Cryo‐electron Microscopy to Discover Box C/D s(no)RNP Structure
Authors: Baserga Susan J , Yip W.S. Vincent , Shigematsu Hideki , Taylor David W. 
Published: November 2023

Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′‐O‐methylation, one type of rRNA modification found in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs from our laboratory have demonstrated the dimeric sRNP (di‐sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Because of the limitations of the structural techniques, the orientation of the box C/D sRNAs in the di‐sRNP has remained unclear. Here, we have used cryo‐EM to elucidate the orientation of the sRNAs in a M. jannaschii box C/D di‐sRNP. The cryo‐EM reconstruction suggests a parallel orientation of the two sRNAs. This is further confirmed by results from biochemical and structural analyses of sRNPs assembled with mutant sRNAs that indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di‐sRNP architecture.Support or Funding InformationSupported by NIH R01GM0115710
Functional Significance of Intermediate Cleavages in the 5′ETS of Schizosaccharomyces pombe pre‐rRNA
Authors: Van Loggerenberg Warren , Nellimarla Srinivas , Park James , Nazar Ross N. 
Published: November 2023

The biosynthesis and incorporation of mature ribosomal RNAs (18S, 5.8S and 25S rRNA) into ribosomes is dependent on coordinated and interdependent processing events, acting as a “quality control mechanism”. Transcribed spacers are critical to processing and act as “biological springs” to enable the appropriate configuration of maturing termini. The pre‐rRNA 5′ external transcribed spacer (5′ ETS) of Schizosaccharomyces pombe contains structural elements critical for initiation and efficient pre‐rRNA processing. Examination of termini in the 5′ ETS region based on ligation‐mediated RT‐PCR revealed two major intermediate cleavage sites (A1 and A2) in addition to the A0 cleavage, which forms the mature 5′ end of the 18S rRNA. Mutational analyses at the A2 intermediate cleavage site indicate that this cleavage is not critical to rRNA maturation and can be eliminated. Mapping of the 5′ ETS region in cells with temperature sensitive nucleases, using RT‐PCR, refute formation of the mature 5′ end of 18S rRNA by RNase III‐like endonucleolytic cleavage across an extended hairpin. Mutational analysis around the 5′ ETS/18S rRNA junction indicate cleavage is not dependent on sequence and/or structure, but can dramatically affect rRNA yield. Additionally, mutations in U3 snoRNA or a complementary site in the 5′ 18S rRNA pseudoknot can significantly affect the 18S/25S rRNA ratio. These results raise the possibility that the 5′ end of the 18S rRNA is the result of ribozyme cleavage and that the U3 snoRNA acts as a chaperone to stabilize RNA structure.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada.
BDF2 Transcript Sensitivity to RNase III‐Mediated Decay is Heavily Governed by its Transcript Localization and Vulnerability to Cleavage
Authors: Wang Charles , Roy Kevin , Chanfreau Guillaume 
Published: November 2023

Bromodomain factors are emerging as key players in transcription regulation through chromatin remodeling and transcription initiation. Due to their significant impact on the eukaryotic transcriptome, they are often under the control of several regulatory mechanisms to ensure the appropriate modulation of gene expression. One such factor in yeast, Bromodomain Factor 2 (BDF2), has been established as a target of both RNase III‐mediated decay (RMD) and spliceosome‐mediated decay (SMD) for extensive surveillance. Although our previous work has shown that the RMD pathway for the BDF2 transcript is hyper‐activated by salt stress and other environmental conditions, the mechanism driving the enhanced cleavage of BDF2 RNA under these conditions remains obscure. Through the anchor‐away technique, we show that inhibiting the export of the BDF2 transcript out of the nucleus can recapitulate the stress‐induced RMD hyper‐activation. In addition, we also show in vitro and in vivo that the cleavage efficiency of the RNase III (Rnt1p) on BDF2 may further contribute to its vulnerability to RMD. Altogether, these results suggest that the activity of RMD is heavily governed by the localization of its target transcripts, and that the efficiency of cleavage may further contribute to the sensitivity of these transcripts to degradation.Support or Funding InformationCellular and Molecular Biology Training Grant
Structural and Functional Characterization of a Human Ribonuclease Implicated in Obesity
Authors: Abshire Elizabeth , Chasseur Jennifer , Bohn Jennifer , DelRizzo Paul , Goldstrohm Aaron , Trievel Raymond 
Published: November 2023

Obesity affects a significant number of Americans and greatly increases the risk of developing cardiovascular disease, type‐II diabetes, fatty liver disease, and certain cancers. Understanding how obesity is regulated opens up new avenues of pharmacological interventions to treat obesity, concurrently reducing the risk of co‐morbidities. Mice lacking the broadly expressed enzyme Nocturnin (NOCT) are resistant to diet‐induced obesity and other studies have observed a role for this enzyme in regulating intestinal trafficking of dietary fats and promoting adipogenesis. We have characterized human NOCT and determined that it represses the activity of reporter RNAs in tethered function assays promotes decay of reporter RNAs in vivo. These observations are consistent with a proposed function for NOCT in RNA decay and suggest that there may be a novel pathway that regulates lipid metabolism by targeting mRNAs for degradation. Though the broader physiological effects of gene deletion of NOCT have been described, discovering its molecular function remains an important goal. We have utilized structural and approaches to determine the first crystal structure of the human homolog of NOCT to aid in elucidating its catalytic mechanism. Additionally, we have developed cell‐based assays that will be used to study NOCT function and analyze the effects of active site mutants. These studies describe the structure and activity of human NOCT and provide the foundation for characterizing this enzyme as a potential drug target for obesity and related co‐morbidities.Support or Funding InformationNational Institutes of Health Chemistry‐Biology Interface Training Program (NIH grant 5T32GM008597), American Heart Association Midwestern Affiliate Predoctoral Fellowship, Michigan Nutrition and Obesity Research Center Pilot Grant
Investigating the Role of the Human Parvovirus B19's Main Viral Protein, NS1, in Viral Replication and Interactions with Host DNA
Authors: Hernandez Christian Joab , Sanchez Jonathan Lares , Horton Nancy C. 
Published: November 2023

Human Parvovirus B19 (B19V) is a ubiquitous virus that infects the majority of the human population. B19V is a non‐enveloped, single stranded DNA virus with a viral genome composed of 5594 nucleotides. Infection with B19V has been associated with a multitude of diseases, such as hydrops fetalis, arthropathy, hepatitis, cardiomyopathy, erythema infectiosum (Fifth disease), and possible onset of a variety of autoimmune diseases. The viral genome of B19V encodes for five known, possible six, protein products: VP1 and VP2 that compose the capsid, NS1, the main viral protein, and two to three smaller protein products of indistinct function. The main replicative protein, NS1, is a multiple domain protein with a nuclease domain, helicase domain, and a C‐terminal domain. Through its multi‐functional capabilities, NS1 has been hypothesized to play several roles in the triggering autoimmune disease via formation of covalent DNA‐NS1 adducts and/or host gene transactivation. In order to understand the mechanism behind NS1's ability to bind viral and host genomic DNA, biochemical experiments were carried out to build a model for NS1's role in viral genome replications and host gene transactivation capabilities.Support or Funding InformationSupported by: the National Science Foundation under Grant MCB1410355, and National Institutes of Health via Grant S10OD013237
Recruitment of Drp1 in Mitochondrial Fission
Authors: Egner John M , Bakkum Amber , Gardner Alex , Hill Blake 
Published: November 2023

Intracellular membranes are dynamically shaped by proteins including fission and fusion events important for vesicular transport, organelle trafficking, and cellular homeostasis. Yet how proteins interact with membranes to control these events is largely unknown. We are interested in addressing this question through mechanistic studies of a dynamin superfamily member central to the division, or fission, of mitochondria. Mitochondrial fission is driven by the GTPase mechanoenzyme, Dynamin‐related protein 1 (Drp1). At the mitochondrial surface, Drp1 is thought to assemble into rings around a future site of scission in a GTP‐dependent manner. Curiously, Drp1 is primarily localized to the cytoplasm and how it is recruited to the mitochondrial surface is unknown. Several proteins resident to the mitochondrial surface have been implicated in Drp1 recruitment and subsequent assembly, but the mechanism remains unclear. We are addressing these problems using biochemical, structural, and cell biological approaches. Our results to date will be presented and suggest a new model for Drp1 activity in mitochondrial fission.Support or Funding InformationThis project was supported by the Clinical & Translational Science Institute (CTSI) through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Project also supported by NIH grants NIH R01‐GM067180, and NIH R01‐HL128240.
Molecular Chaperones of the Hsp70 Family Interacting with the Proteasome Assembly Network
Authors: Hammack Lindsay J , Firestone Kyle , Kusmierczyk Andrew R 
Published: November 2023

Proteins Ssa1 and Ssa2 are members of the HSP70 family. These nearly identical ATPases are well characterized in maintaining protein homeostasis. Their functions include aiding in protein folding and targeting short‐lived proteins for degradation. These proteins are highly abundant and are routinely detected in mass spectrometry data following affinity purification. However, due to their abundance, these proteins are often assigned to groups of proteins that non‐specifically interact with the purification matrix. Perhaps the best known of these collections is the CRAPome, an online repository of common contaminants found in affinity purification. Even though Ssa1 and Ssa2 are part of the CRAPome, we find that Ssa1/Ssa2 interact with assembling proteasomes, specifically with defined subcomplexes of the core particle. Furthermore, our genetic results also indicate that Ssa1/Ssa2 interact with the with the proteasome assembly network. Proteasome assembly has been characterized as a stepwise process that includes dedicated proteasome assembly chaperones. This work supports an active role for the general molecular chaperones, Ssa1/Ssa2, in this process as well.Support or Funding InformationThis work was funded in part by a Research Support Funds Grant from IUPUI (to A.R.K.).
ScMC2 is a Calcium‐Dependent Metacaspase Cleaving Peptides after Arg Residues
Authors: Scharbach Stephanie N , Fox Kristin 
Published: November 2023

Programmed cell death (PCD) is a terminal pathway important in the maintenance of cell homeostasis. Recent studies have shown that PCD can also happen in unicellular organisms. Like all pathways in the cell, PCD must be controlled. Metacaspases have been implicated in the cell death pathway in yeast and are present only in protozoa, fungi and plants. Metacaspases are proteases which are cysteine‐dependent and distantly related to the caspases found in metazoans. Some metacaspases require the presence of calcium for activation. Most caspases have a prodomain that must be cleaved to produce active enzyme.This project focuses on characterizing Schizophyllum commune metacaspase 2 (ScMC2). To clone ScMC2 with and without the prodomain, the genes were amplified using PCR and fused to small ubiquitin modifier (SUMO) in a vector via Gibson Assembly. ScMC2 and ScMC2Δpro were then expressed and purified using nickel affinity chromatography.ScMC2 and ScMC2Δpro expressed using these plasmids were found to be active from the activity studies using the substrate Z‐GRR‐AMC. The optimal concentration of calcium in enzyme assays for MC2 was determined to be 25 mM. This is higher than the optimal concentration for the other S. commune metacaspases. ScMC2 and ScMC2Δpro were successfully cloned and when expressed produced an active metacaspase.