bluuebunny/crossref_metadata_2025_split huggingface dataset:
The RS/RGG Domain of the SR‐like Protein Npl3 is Required for Efficient RNA Splicing in Yeast
Authors: Lin Mengjia M , Gala Kinisha , Lobby Jenna , Moazamian Aron , Kress Tracy L 
Published: June 2021

Npl3 is an SR‐like protein found in Saccharomyces cerevisiae that functions in co‐transcriptional pre‐mRNA splicing by promoting the recruitment of splicing factors to the pre‐mRNA during transcription1. It contains two RNA recognition motifs (RRMs), an RS/RGG domain rich in arginine and serine repeats, and an uncharacterized N‐terminus. While Npl3 binds RNA2–4 via the RRM domains5, the role of each of the functional domains in splicing is currently unknown. Here we have generated a panel of flag‐tagged Npl3 proteins lacking one or more of the protein domains. We identified genetic interactions between Npl3 mutations lacking the RS/RGG domain and mutations in genes that encode RNA splicing factors, suggesting that the RS/RGG domain is important for RNA splicing. Indeed, we show that deletion of the RS/RGG domain of Npl3 causes a significant decrease in RNA splicing efficiency of Npl3‐dependent pre‐mRNAs. In addition, deletion of the RS/RGG domain abolishes the ability of Npl3 to interact with splicing factors, which strongly suggest that the RS/RGG domain is required to recruit the splicing machinery during transcription. Interestingly, we found that phosphorylation of the RS/RGG domain is not important for RNA splicing. Replacing the Npl3 RS/RGG domain with the RS/RGG domain from the Candida albicans Npl3 ortholog6 restores growth, interaction with splicing proteins, and RNA splicing. Together this work demonstrates that the RS/RGG domain is required for the activity of Npl3 in RNA splicing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the ERK/MAP kinase long first intron and its possible role in gene regulation and germ cell development
Authors: McCloskey John , Robinson‐Thiewes Sarah , Kimble Judith 
Published: June 2021

One distinguishing feature of eukaryotic genomes is the presence of introns within nascent transcripts. Genomic analyses of distantly related species, spanning yeast to humans, reveal conservation of intron size and position, suggesting an intron can be advantageous. Many studies have examined the relationship between short introns and gene expression; however, the effect of long introns and gene expression is not well understood. Critical developmental regulators often have an unusually long intron that is conserved in size and position and typically one of the first introns. We hypothesize that long first introns of developmental regulators affect gene expression. To investigate the relationship between a long first intron and gene expression, we have focused on the Caenorhabditis elegans gene encoding ERK/MAP kinase (mpk‐1). Like other metazoan ERK/MAP kinases, mpk‐1 harbors a long first intron in one of its isoforms. The mpk‐1b nascent transcripts include a small first exon and an 8.2 kb first intron, while mpk‐1a lacks that first exon and intron but shares other exons and introns with mpk‐1b. To visualize RNAs, we designed single molecule in situ hybridization (smFISH) probes targeting the 8.2 kb intron to visualize nascent transcripts as well as other probes targeting the shared exons to detect the mRNAs. We use confocal microscopy and MATLAB image processing to assess mpk‐1 transcription from germline stem cell self‐renewal to meiotic pachytene of differentiating cells. In parallel, we are using CRISPR to delete regions of the long mpk‐1b intron to assess its functional importance. Our plan is to compare mpk‐1 nascent and mature transcripts during germline development, both in wild type and in in mutants that lack part of most of that large intron. Our analysis will tell us if the mpk‐1b 8.2 intron is critical for the mpk‐1 regulatory program during germ cell development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the molecular mechanism of ProQ‐RNA interactions using a bacterial three‐hybrid assay
Authors: Pandey Smriti , Gravel Chandra , Berry Katherine 
Published: June 2021

In bacteria, small RNAs (sRNAs) play important roles in gene regulation; sRNAs can regulate the translation and stability of target mRNAs via imperfect base pairing. These sRNA‐mRNA interactions are often facilitated by RNA chaperone proteins. ProQ has recently been identified as a global RNA‐binding protein that binds to dozens of sRNAs and hundreds of mRNAs in multiple proteobacteria; this observation has led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression.Our goal is to understand the molecular mechanisms of ProQ's interaction with regulatory RNAs, mapping the amino acids on ProQ's surface and nucleotides of RNAs that contribute to binding and regulation by using a bacterial three‐hybrid (B3H) assay to genetically detect ProQ‐RNA interactions. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing either an sRNA or 3′UTR of interest is tethered to a DNA sequence upstream of a test promoter. Interaction of ProQ with the RNA stabilizes the binding of RNAP to the test promoter and activates transcription of a reporter gene.We have detected preliminary B3H interactions of ProQ with several of its RNA partners. Our data supports a model where the conserved N‐terminal‐domain (NTD) is the primary region for RNA binding. Further, we have identified point mutations in ProQ that affect its RNA interactions and have used immunodetection to determine ProQ expression levels to ensure that these mutations are not destabilizing. Current efforts are focused on screening for additional ProQ point mutations to locate the binding interface(s) for the sRNAs and mRNAs with which it interacts as well as exploring the structure and sequences of RNA that are required for ProQ interaction.Support or Funding InformationResearch supported by Mount Holyoke College and the Luce FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA binding properties of the Lsm1–7 ring from Schizosaccharomyces pombe
Authors: Virta Johanna M , Hayes Samuel M , Montemayor Eric J , Butcher Samuel E 
Published: June 2021

Lsm proteins form heptameric rings that bind RNA. Eukaryotes contain Lsm1–7 and Lsm2–8 ring structures, which are comprised of Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7 and either Lsm1 or Lsm8. Thus, the two rings differ by only one component. This small change in composition has a profound effect on the biological function of the two rings. Lsm1–7 is located in the cytosol and plays an essential role in the decay of messenger RNA, whereas Lsm2–8 is located in the nucleus and specifically binds to the 3′ end of U6 RNA. It is important to understand the RNA binding properties of Lsm rings. For example, the Lsm1–7 mRNA decay pathway regulates a large proportion of gene expression (Garneau et al., 2007).It is known that Lsm2–8 binds to U6 snRNA via a 3′ polyuridine stretch and a terminal phosphate group (Licht et al., 2008; Didychuk et al., 2017). In contrast, the RNA binding properties of the Lsm1–7 ring are not well understood. Free Lsm1–7 rings in S. cerevisiae lack tight binding affinity for polyuridine tracts (Zhou and Zhou, et al., 2014). The cognate RNA sequence for the free Lsm1–7 ring is unknown, although association of the ring with the Pat1 protein can stimulate binding of the ring to polyuridine tracts (Wu et al., 2014). Crystal structures of the free Lsm1–7 complex from S. cerevisiae show that the C‐terminal region of Lsm1 impinges on the uridine‐phosphate binding pocket observed in the S. cerevisiae Lsm2–8 ring with U6 snRNA (Zhou and Zhou, et al., 2014; Sharif and Conti, 2013; Montemayor et al., 2018). We hypothesized that Lsm1 is a central regulator of RNA binding activity in the Lsm1–7 complex and that Pat1 is an allosteric regulator of Lsm1.We have purified Lsm rings from the fission yeast S. pombe, which closely resemble humans with respect to Lsm ring sequence and known binding properties. We have made several novel observations. First, free Lsm1–7 is capable of binding polyuridine tracts in the absence of the Pat1 cofactor if the polyuridine tract is followed by an adenosine residue. This is the first observation of tight RNA binding affinity for Lsm1–7. Second, we find that deletion of the C‐terminal region of Lsm1 allows Lsm1–7 to bind polyuridine tracts 10‐fold more tightly in the absence of Pat1 or the single adenosine residue. These data support our hypothesis that the C‐terminal region of Lsm1 inhibits association of the ring with RNA, and as such is a negative regulator of RNA binding activity. Finally, we were able to determine a 1.9 Å resolution crystal structure of the S. pombe Lsm1–7 ring (lacking the C‐terminal region of Lsm1) in complex with a polyuridine RNA. This structure shows that the uridine nucleotides bind into the same binding pockets as in Lsm2–8, as hypothesized previously (Montemayor et al., 2018). This work expands our understanding of how the Lsm1–7 complex binds RNA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RaPID Analysis of Cytomegalovirus of Long Noncoding RNA Binding Proteins
Authors: Lee Jessica E. , Kulesza Caroline 
Published: June 2021

Human Cytomegalovirus (HCMV) is a ubiquitous herpes virus that is widespread and usually asymptomatic among healthy individuals. Congenital HCMV infections are the leading cause of virally induced birth defects. HCMV also causes severe disease in immunocompromised patients. Previous work in our laboratory has shown that CMV encodes a large noncoding RNA called RNA5.0. Using an animal model, the homologous RNA in the murine model called RNA7.2 has been shown to help the virus persist in host salivary glands, but it is unclear how this works at the molecular level. To identify the proteins attaching to RNA5.0, the RaPID system will be used. The RaPID system uses a highly active biotin ligase that tags proteins which bind to specific RNA sequences in cells. The RaPID system was pioneered in Paul Khavari's lab to demonstrate a small cellular RNA (EDEN15) binds to the CELF1 protein. We have reproduced their data in our laboratory and will use this system to identify RNA5.0 binding proteins. We will be looking for proteins that bind to the 3′ end of RNA5.0 because there is evidence this sequence is related to processing and stability of the RNA. Moving forward different length fragments spanning the 3′ end of RNA5.0 will be used in the RaPID system to identify which proteins interact with the sequence. The identified proteins might point out specific pathways to target for a potential treatment for CMV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncovering Caprin1's biological role to understand it's function in autism
Authors: Martinez Criseyda , Pani Bibhusita , Nudler Evgeny 
Published: June 2021

Deregulation of various cellular pathways such as the immune, mRNA translation and cell cycle pathways have been linked to autism disorders, yet the cause of deregulation remains unclear. Here we study the Cytoplasmic Activation/Proliferation‐Associated Protein‐1 (Caprin1), an RNA binding protein implicated in autism. In neurons, Caprin1 binds and represses the translation of selective mRNAs at RNA granules. Upon various stimulations such as with brain derived neurotropic factor (BDNF), mRNAs dissociate from Caprin1 and shift into actively translating ribosomes. In addition, studies identified Caprin1 as a positive cell cycle regulator, allowing cells to transition between G1 and S phase by binding to upregulated cell cycle mRNAs. Yet, how Caprin1 mediates repression or promotion of different mRNAs is not well understood. To identify and understand the global mechanism of Caprin1 we conducted a series of high throughput assays including mass‐spectrometry‐based quantitative proteomics and RNAseq. We show that in the absence of Caprin1, cell cycle and cell metabolic genes were significantly decreased. On the other hand, absence of Caprin1 significantly increased genes involved in immune responses, including antigen processing. To further identify a mechanism we conducted a crosslinking‐immunoprecipitation (CLIP) assay to identify mRNAs bound directly to Caprin1. We find that Caprin1 mainly binds to mRNAs upstream of cytokine signaling and cell cycle pathways at the 3′UTR. We further show that Caprin1 degrades or stabilizes different bound mRNAs. Thus, we conclude that Caprin1 has a role in RNA stability, particularly in genes involved in cell cycle and immune response pathways, which are deregulated and implicated in autism.Support or Funding InformationRobertson Foundation, NIH R01AI090110 Supplement, Howard Hughes Medical Institute (HHMI)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Tat‐specific Factor 1 Binds and Exports HIV‐1 RNA to the Cytoplasm
Authors: Miller Heather 
Published: June 2021

Human immunodeficiency virus type 1 (HIV‐1) requires many human cofactors for propagation. Multiple groups have shown that human Tat‐specific factor 1 (Tat‐SF1) is an HIV‐1 dependency factor. This protein's knockdown does not affect the overall levels of viral RNA, however it does regulate the relative size classes (unspliced, singly spliced, and fully spliced). The molecular mechanism behind this is unknown, but data is consistent with roles in enhancing splicing, export, and/or stability of the viral RNA. We hypothesized that if Tat‐SF1 plays any one of these roles, then we should be able to demonstrate binding of the protein to the RNA genome. Furthermore, if Tat‐SF1 is critical for nuclear export of the viral RNAs, then knocking down Tat‐SF1 should result in more nuclear retention of the viral genome. Fragments of the HIV‐1 genome were synthesized by in vitro transcription and end‐labelled with fluorescein. These RNA probes were used in both electrophoretic mobility shift assays (EMSA) and florescence polarization (FP) assays. To analyze RNA export, HeLa cells with an shRNA‐expressing plasmid targeting Tat‐SF1 were also transfected with pSG3Δenv, a non‐infectious HIV‐1 plasmid. RNA from nuclear and cytoplasmic fractions was purified, followed by RT‐qPCR analysis. Our results suggest that Tat‐SF1 plays a role in binding the HIV‐1 genome and exporting the unspliced transcripts to the cytoplasm. This work provides additional molecular details on how Tat‐SF1 acts as an HIV‐1 dependency factor.Support or Funding InformationHigh Point UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Stress Modulates Elongator Protein 3 (ELP3) Function to Enhance Dopaminergic Neurotoxicity in Cell Culture and Animal Models of Parkinson's Disease
Authors: Abdalla Ahmed , Charli Adhithiya , Ghaisas Shivani , Kondru Naveen , Malovic Emir , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Kanthasamy Anumantha 
Published: June 2021

Mitochondrial dysfunction is well established in Parkinson's disease (PD). Exposure to mitochondria‐targeting neurotoxicants, e.g., those neurotoxic pesticides that act as mitochondrial complex I inhibitors, has been linked to the pathogenesis of environmentally linked PD, but the molecular mechanisms underlying the enhanced vulnerability of dopaminergic neurons to mitochondrial toxicants are not fully understood. The histone acetyltransferase ELP3 (Elongator Protein 3) is the catalytic subunit of the RNA polymerase II elongator complex and plays an essential role in transcript elongation and maturation of projection neurons. Dysregulation of ELP3 has been implicated in a number of human disorders, but its potential role in the pathogenesis of PD remains elusive. In a serendipitous finding, we observed that mitochondria‐targeting pesticides can disrupt ELP3's function in dopaminergic neurons in cell culture and rodent models of PD. In this study, we further characterized ELP3 expression in the mouse brain and found high expression of full‐length ELP3 (62 kDa) in cytosolic fractions from different brain regions, while high levels of short‐form ELP3 (~40 kDa) were detected in the olfactory bulb (OB). Interestingly, immunohistochemical analyses revealed that ELP3 is highly expressed in TH‐positive neurons of the OB. Furthermore, OB slice cultures treated with the mitochondrial complex‐I inhibiting pesticide tebufenpyrad (Tebu) showed a reduced level of short‐form ELP3 in mitochondrial fractions. Consistently, we also found that ELP3 levels were significantly downregulated in the OB of transgenic MitoPark mice, a model which recapitulates mitochondrial dysfunction in PD. Collectively, our results indicate that ELP3 is a novel cellular target that can modulate the susceptibility of dopaminergic neurons to neurotoxic mitochondria‐targeting stressorsSupport or Funding InformationNIH grants ES026892 and ES027245 and Eugene and Linda Lloyd Endowed ChairThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Acetylation on Beta‐2‐Microglobulin on Amyloid Formation
Authors: Ballard Abigail Rene , Richardson John 
Published: June 2021

Beta‐2‐Microglobulin (B2m) is a small 99 residue globular protein that is a part of the type I major histocompatibility complex found on all nucleated cells. It is shed from the surface of cells into the bloodstream as cells undergo apoptosis then is degraded and excreted from the body in people with normal functioning kidneys. However, B2m is not effectively removed from the blood by hemodialysis causing systemic problems for patients in end‐stage kidney disease (ESKD). B2m serum levels in patients with ESKD are up to 60 times greater than healthy individuals. Dialysis related amyloidosis (DRA), is a disease that is often experienced by those with ESKD. DRA results from the accumulation of misfolded B2m and is characterized by carpal tunnel like symptoms coupled with chronic joint inflammation. DRA is a result of the deposition of misfolded B2m in the musculoskeletal system due to B2m amyloid plaque formation. However, the increased concentration of B2m alone is not sufficient to trigger the pathogenic amyloidosis at physiological pH. Thus, a secondary factor must be acting as the trigger of misfolding. This study is investigating the effect of acetylation as a non‐natural post translational modification on B2m. Acetylation is a practicable suspect as aspirin consumption is known to acetylate a variety of proteins non‐specifically. The acetylation of lysines would alter the overall charge and possibly disrupt ability of B2m to maintain its native state conformation thus increasing the aggregation propensity of B2m. We are primarily testing the susceptibility of B2m to acetylation via protocol established for acetylation in bovine carboxy anhydrase. Additionally, growth of the amyloid fibrils of the acetylated B2m will be monitored with thioflavin‐T fluorescence to assess the effects of lysine acetylation on B2m amyloid formation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D‐derived peptide inhibitor
Authors: YANG JIE , Liu Zhonghua , Wang Chuanping , Yang Rui , Rathkey Joseph K. , Shi Wuxian , Chen Yinghua , Dubyak George R. , Abbott Derek W. , Xiao Tsan Sam 
Published: June 2021

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases‐1/4/5/11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N‐terminal domain to associate with membrane lipids and form pores, which induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full‐length GSDMD and its cleavage site peptide FLTD. A GSDMD‐derived inhibitor N‐Acetyl‐Phe‐Leu‐Thr‐Asp‐Chloromethylketone (Ac‐FLTD‐CMK) inhibits GSDMD cleavage by caspases‐1/4/5/11 in vitro, suppresses pyroptosis downstream of both canonical and non‐canonical inflammasomes, as well as reduces IL‐1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase‐3 or apoptotic cell death, suggesting that Ac‐FLTD‐CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase‐1 in complex with Ac‐FLTD‐CMK reveals extensive enzyme‐inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase‐1 structures demonstrates drastic conformational changes at the four active‐site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases, which is a novel tool for investigating inflammasome signaling.Support or Funding InformationT.S.X. is supported by NIH grant AR069908 and the Case Research Institute. Z.L. is supported by a postdoctoral fellowship from the American Association of Immunologists. J.K.R. is supported by NIH grant T32 GM007250. D.W.A. is funded by NIH grants DK091222 and GM086550.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.