Genetics and Genomics Commons^™

Mitochondrial Trna-Derived Fragments As Candidate Metastasis-Modifying Rna, Katy L. Swancutt, R. Mckinnon Walsh, Sydney Quijano, Emily Schueddig, Devin C. Koestler, Adam D. Scheid, Tony Vanden Bush, Yi Jing, Isidore Rigoutsos, Danny R. Welch

Computational Medicine Center Faculty Papers

UNLABELLED: How mitochondrial DNA (mtDNA) polymorphisms influence complex phenotypes remains poorly understood. Using mitochondrial-nuclear exchange mice, we previously showed that mtDNA single-nucleotide polymorphisms (SNP) modify metastasis, cardiovascular disease, and epigenetic marks independently of metabolic differences. The only mtDNA SNP correlating with these phenotypes resides in the gene encoding mitochondrial transfer RNA (tRNA)-arginine [mt-tRNAArg (UCG), mt-TR], suggesting a role for non-protein-coding loci. In this study, we identify and preliminarily characterize previously undescribed tRNA-derived fragments (tRF) generated from mt-TRs. Northern blotting revealed distinct tRF that are differentially expressed among mtDNA SNPs, between lung and liver, and between sexes. Surprisingly, small RNA sequencing …

Integrative Rna-Seq Analysis Reveals Stress Type-Dependent Lncrna-Centered Co-Expression Networks Across Human Cellular Stress Contexts, Christina Anastasiadi, Aggeliki Kasapi, Ioannis Sentementes, Vasileios Gouzouasis, Margaritis Tsifintaris, Antonis Giannakakis

Computational Medicine Center Faculty Papers

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular adaptation to environmental and molecular stress, but the extent to which their responses remain reproducible and stress-type-dependent across human stress conditions remains unclear. Here, we performed an integrative transcriptomic meta-analysis of human stress-response datasets from ASTRA and GEO, focusing on normal, non-cancerous, wild-type human cell lines exposed to oxidative stress (H2O2), hypoxia, heat stress, or UV-induced DNA damage. Gene Ontology (GO) enrichment analysis of differentially expressed (DE) protein-coding mRNAs confirmed that the resulting stress-stratified comparison captured biologically coherent transcriptional programmes to oxidative stress signaling, hypoxic and metabolic adaptation, heat-induced …

Isolation And Quantification Of Rna From Extracellular Vesicles Derived From A549 Human Non-Small Cell Lung Cancer Cells Using Ultracentrifugation, Yousef Dimian, Vanessa Pizutelli, Dimitri Pestov

Rowan-Virtua Research Day

Extracellular vesicles (EVs) have gained attention as potential diagnostic tools due to their presence in body fluids and their ability to carry nucleic acids, including RNA. EV-derived RNA, particularly ribosomal RNA (rRNA), may reflect cellular stress and disease states, making it a promising biomarker1, 2. This project focuses on optimizing methods to isolate EVs and quantify their RNA content using human cell culture models. Differential ultracentrifugation was used to separate vesicle populations based on size, followed by RNA extraction and quantification using a high-sensitivity assay. Initial experiments using media from three 150 mm culture dishes yielded no detectable RNA, whereas …

Precursor Rna Properties Driving Cryptic 3’ Splice Site Selection In Sf3b1 Mutant Malignancies, Austin A. Herbert

All Dissertations

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3’ splice site selection. SF3B1 is commonly mutated in myelodysplastic syndromes and other blood cancers with the K700E mutation being the most frequent. This mutation of SF3B1 induces cryptic 3’ splice site activation in hundreds of genes. Introduction of such cryptic 3’ splice sites can shift the reading frame of protein coding transcripts, causing the transcript to be tagged for nonsense-mediated decay or resulting in the production of a truncated protein. Both these scenarios can lead to down-regulated protein expression. Sequence and structural properties of precursor …

Organism-Specific Sequence Motifs Link Ribosomal Rnas To Brain Disorders, Isidore Rigoutsos, Stepan Nersisyan, Eric Londin, Iliza Nazeraj, Bonnie Dong, Anastasios Vourekas, Phillipe Loher

Computational Medicine Center Faculty Papers

We report that in humans, mice, fruit flies, and worms, the ribosomal RNAs and the transcribed spacers of 45S are densely packed with organism-specific sequence motifs that are primarily shared with nervous system genes. The human ribosomal RNAs and 45S spacers contain 1,723 such motifs. Specific combinations of these motifs are predominantly found in 3,430 human nervous system genes, of which 1,046 are genes associated with brain disorders, including autism spectrum disorder and schizophrenia. The sequences of the 1,723 motifs and their locations in the introns and exons of nervous system genes are unique to primates. Experimental evidence indicates that …

Development Of Emerin Mrna Lipid Nanoparticles To Rescue Myogenic Differentiation., Nicholas Marano, Liza Elif Guner, Rachel S Riley, James M Holaska

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Emery-Dreifuss muscular dystrophy 1 (EDMD1) arises from mutations in EMD. Most EDMD1 patients lack detectable emerin expression. They experience symptoms such as skeletal muscle wasting, joint contractures, and cardiac conduction defects. Currently, physicians rely on treating patient symptoms without addressing the underlying cause-lack of functional emerin protein. Thus, there is a need for therapeutic approaches that restore emerin protein expression to improve patient outcomes. One way would be to deliver emerin mRNA or protein directly to affected tissues to restore tissue homeostasis. Here, we evaluated the utility of lipid nanoparticles (LNPs) to deliver emerin mRNA to diseased cells. LNPs …

Hypoxia Induced Ribosomal Rna Fragmentation Mediated By Rnase L, Vanessa Kristina Pizutelli, Dimitri G Pestov

Rowan-Virtua Research Day

Ischemic injury contributes to a range of global pathologies. Ischemia/Reperfusion Injury (IRI) is a paradoxical phenomenon that involves an initial restriction of blood flow followed by a sudden restoration of perfusion. This type of injury takes place in conditions such as myocardial infarction, acute kidney disease, and ischemic stroke and often leads to cellular death. Additionally, IRI exacerbates post-surgical outcomes and plays a role in graft dysfunction and transplant rejection. Despite efforts to develop therapies targeting known IRI pathways, clinical trials have largely been unsuccessful, underscoring the need for alternative mechanisms and biomarkers associated with IRI-induced apoptosis. Reactive oxygen species …

The Role Of Secondary And Tertiary Structure In The Cap-Independent Translation Of Fgf-9 And Hif-1-Alpha, Amanda Michelle Whittaker

Dissertations, Theses, and Capstone Projects

Under normoxic conditions, eukaryotes initiate translation of RNA through eIF4E recognition of the 5’ cap. However, under cellular stress, eukaryotic translation must be initiated through a 4E-independent, or “cap-independent” mechanism, involving eukaryotic initiation factor 4G (eIF4G) binding directly to the 5’ untranslated regions (5’ UTR) of the RNA. eIF4G binding then recruits the ribosome to the transcript. While this mechanism is useful for translation of apoptotic transcripts and transcripts involved in cell survival, cap-independent translation is also utilized by oncogenic RNA for tumorigenesis. Previous work by our lab and others has categorized this recruitment and initiation mechanism as either internal-ribosome-entry-site …

Mitochondrial Mrna And The Small Subunit Rrna In Budding Yeasts Undergo 3'-End Processing At Conserved Species-Specific Elements, Michael Anikin, Michael F Henry, Viktoria Hodorova, Hristo B Houbaviy, Jozef Nosek, Dimitri G Pestov, Dmitriy A Markov

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include

The Importance Of Solution Studies For The Structural Characterization Of The Enterovirus 5' Cloverleaf, Morgan G. Daniels, Meagan E. Werner, Xiaobing Zuo, Steven M. Pascal

Chemistry & Biochemistry Faculty Publications

Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5' cloverleaf (5'CL). Here, we present a biophysical analysis of the 5'CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5'CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed …

Codon Composition In Human Oocyters Reveals Age-Associated Defects In Mrna Decay, Pavla Brachova, Lane K. Christenson, Nehemiah S. Alvarez

Department of Biomedical and Translational Sciences Faculty Publications

Oocytes from women of advanced reproductive age exhibit diminished developmental potential, but the underlying mechanisms remain incompletely defined. Oocyte maturation depends on translational control of maternal mRNA synthesized during growth. We performed a computational analysis on human oocytes from women < 30 versus ≥40 years and observed that mRNA GC content correlates negatively with half-life in oocytes from young (< 30 yr) but positively with oocytes from aged (>40 yr) women. In young oocytes, longer mRNA half-life is associated with lower protein abundance, whereas in aged oocytes GC content correlates positively with protein abundance. During the GV-to-MII transition, codon composition stratifies stability: codons that support rapid translation (optimal) stabilize mRNA, while slow-translating codons (non-optimal) promote decay. With reproductive aging, GC-containing codons become more optimal and align with increased …

Steering Research On Mrna Splicing In Cancer Towards Clinical Translation, Olga Anczukow, Frédéric H-T Allain, Brittany L Angarola, Douglas L Black, Angela N Brooks, Chonghui Cheng, Ana Conesa, Edie I Crosse, Eduardo Eyras, Ernesto Guccione, Sydney X Lu, Karla M Neugebauer, Priyanka Sehgal, Xiao Song, Zuzana Tothova, Juan Valcárcel, Kevin M Weeks, Gene W Yeo, Andrei Thomas-Tikhonenko

Faculty, Staff and Students Publications

Splicing factors are affected by recurrent somatic mutations and copy number variations in several types of haematologic and solid malignancies, which is often seen as prima facie evidence that splicing aberrations can drive cancer initiation and progression. However, numerous spliceosome components also 'moonlight' in DNA repair and other cellular processes, making their precise role in cancer difficult to pinpoint. Still, few would deny that dysregulated mRNA splicing is a pervasive feature of most cancers. Correctly interpreting these molecular fingerprints can reveal novel tumour vulnerabilities and untapped therapeutic opportunities. Yet multiple technological challenges, lingering misconceptions, and outstanding questions hinder clinical translation. …

De Novo Variants In The Rnu4-2 Snrna Cause A Frequent Neurodevelopmental Syndrome, Yuyang Chen, Ruebena Dawes, Hyung Chul Kim, Alicia Ljungdahl, Sarah L Stenton, Susan Walker, Jenny Lord, Gabrielle Lemire, Alexandra C Martin-Geary, Vijay S Ganesh, Jialan Ma, Jamie M Ellingford, Erwan Delage, Elston N D'Souza, Shan Dong, David R Adams, Kirsten Allan, Madhura Bakshi, Erin E Baldwin, Seth I Berger, Jonathan A Bernstein, Ishita Bhatnagar, Ed Blair, Natasha J Brown, Lindsay C Burrage, Kimberly Chapman, David J Coman, Alison G Compton, Chloe A Cunningham, Precilla D'Souza, Petr Danecek, Emmanuèle C Délot, Kerith-Rae Dias, Ellen R Elias, Frances Elmslie, Care-Anne Evans, Lisa Ewans, Kimberly Ezell, Jamie L Fraser, Lyndon Gallacher, Casie A Genetti, Anne Goriely, Christina L Grant, Tobias Haack, Jenny E Higgs, Anjali G Hinch, Matthew E Hurles, Alma Kuechler, Katherine L Lachlan, Seema R Lalani, François Lecoquierre, Elsa Leitão, Anna Le Fevre, Richard J Leventer, Jan E Liebelt, Sarah Lindsay, Paul J Lockhart, Alan S Ma, Ellen F Macnamara, Sahar Mansour, Taylor M Maurer, Hector R Mendez, Kay Metcalfe, Stephen B Montgomery, Mariya Moosajee, Marie-Cécile Nassogne, Serena Neumann, Michael O'Donoghue, Melanie O'Leary, Elizabeth E Palmer, Nikhil Pattani, John Phillips, Georgia Pitsava, Ryan Pysar, Heidi L Rehm, Chloe M Reuter, Nicole Revencu, Angelika Riess, Rocio Rius, Lance Rodan, Tony Roscioli, Jill A Rosenfeld, Rani Sachdev, Charles J Shaw-Smith, Cas Simons, Sanjay M Sisodiya, Penny Snell, Laura St Clair, Zornitza Stark, Helen S Stewart, Tiong Yang Tan, Natalie B Tan, Suzanna E L Temple, David R Thorburn, Cynthia J Tifft, Eloise Uebergang, Grace E Vannoy, Pradeep Vasudevan, Eric Vilain, David H Viskochil, Laura Wedd, Matthew T Wheeler, Susan M White, Monica Wojcik, Lynne A Wolfe, Zoe Wolfenson, Caroline F Wright, Changrui Xiao, David Zocche, John L Rubenstein, Eirene Markenscoff-Papadimitriou, Sebastian M Fica, Diana Baralle, Christel Depienne, Daniel G Macarthur, Joanna M M Howson, Stephan J Sanders, Anne O'Donnell-Luria, Nicola Whiffin

Faculty, Staff and Students Publications

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of …

Crispr-Cas9 And Cas12a Target Site Richness Reflects Genomic Diversity In Natural Populations Of Anopheles Gambiae And Aedes Aegypti Mosquitoes, Travis C Collier, Yoosook Lee, Derrick K Mathias, Víctor López Del Amo

Faculty, Staff and Student Publications

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would …

Single-Cell Total-Rna Profiling Unveils Regulatory Hubs Of Transcription Factors, Yichi Niu, Jiayi Luo, Chenghang Zong

Faculty, Staff and Students Publications

Recent development of RNA velocity uses master equations to establish the kinetics of the life cycle of RNAs from unspliced RNA to spliced RNA (i.e., mature RNA) to degradation. To feed this kinetic analysis, simultaneous measurement of unspliced RNA and spliced RNA in single cells is greatly desired. However, the majority of single-cell RNA-seq chemistry primarily captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based single-cell RNA-seq method: snapTotal-seq. We benchmark this method with multiple single-cell RNA-seq assays in their performance in kinetic analysis of cell cycle by RNA velocity. Next, with LASSO regression …

Heat Stress Changes The Bovine Methylome And Transcriptome And Investigation Of Two Novel Genetic Defects In Cattle, Rachel Renae Reith

Department of Animal Science: Dissertations, Theses, and Student Research

Heat stress is a major concern for livestock producers due to its negative impact on animal health and productivity. Heat stress does so by altering expression of genes through different regulatory mechanisms such as DNA methylation. Understanding how heat stress alters gene expression will help elucidate the genetic basis of physiological changes as well as identify targets for possible heat stress mitigation. The purpose of the first study was to understand how heat stress alters the adipose and skeletal muscle transcriptomes in zilpaterol-fed Brahman, as zilpaterol improves muscle growth and may mitigate the effects of heat stress. Differential expression and …

Heat Stress Changes The Bovine Methylome And Transcriptome And Investigation Of Two Novel Genetic Defects In Cattle, Rachel Renae Reith

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Heat stress is a major concern for livestock producers due to its negative impact on animal health and productivity. Heat stress does so by altering expression of genes through different regulatory mechanisms such as DNA methylation. Understanding how heat stress alters gene expression will help elucidate the genetic basis of physiological changes as well as identify targets for possible heat stress mitigation. The purpose of the first study was to understand how heat stress alters the adipose and skeletal muscle transcriptomes in zilpaterol-fed Brahman, as zilpaterol improves muscle growth and may mitigate the effects of heat stress. Differential expression and …

A Single Cell Rna Sequence Atlas Of The Early Drosophila Larval Eye, Komal Kumar Bollepogu Raja, Kelvin Yeung, Yumei Li, Rui Chen, Graeme Mardon

Faculty, Staff and Students Publications

The Drosophila eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. Here, we report transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. Our data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as …

Maldi Imaging Mass Spectrometry Visualizes The Distribution Of Antidepressant Duloxetine And Its Major Metabolites In Mouse Brain, Liver, Kidney, And Spleen Tissues, Saleh M Khalil, Xuan Qin, John M Hakenjos, Jian Wang, Zhaoyong Hu, Xinli Liu, Jin Wang, Mirjana Maletic-Savatic, Kevin R Mackenzie, Martin M Matzuk, Feng Li

Faculty, Staff and Student Publications

Imaging mass spectrometry (IMS) is a powerful tool for mapping the spatial distribution of unlabeled drugs and metabolites that may find application in assessing drug delivery, explaining drug efficacy, and identifying potential toxicity. This study focuses on determining the spatial distribution of the antidepressant duloxetine, which is widely prescribed despite common adverse effects (liver injury, constant headaches) whose mechanisms are not fully understood. We used high-resolution IMS with matrix-assisted laser desorption/ionization to examine the distribution of duloxetine and its major metabolites in four mouse organs where it may contribute to efficacy or toxicity: brain, liver, kidney, and spleen. In none …

An Unexpected Path For Malat1 In Neurons: Trafficking Out Of The Nucleus For Translation, Bradley W Wright, Jeremy E Wilusz

Faculty, Staff and Students Publications

The Malat1 (metastasis-associated lung adenocarcinoma transcript 1) long noncoding RNA is highly and broadly expressed in mammalian tissues, accumulating in the nucleus where it modulates expression and pre-mRNA processing of many protein-coding genes. In this issue of Genes & Development, Xiao and colleagues (doi:10.1101/gad.351557.124) report that a significant fraction of Malat1 transcripts in cultured mouse neurons are surprisingly exported from the nucleus. These transcripts are packaged with Staufen proteins in RNA granules and traffic down the lengths of neurites. They then can be released in a stimulus-dependent manner to be locally translated into a microprotein that alters neuronal gene …

The Trnaval Half: A Strong Endogenous Toll-Like Receptor 7 Ligand With A 5′-Terminal Universal Sequence Signature, Kamlesh Ganesh Pawar, Takuya Kawamura, Yohei Kirino

Computational Medicine Center Faculty Papers

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5'-half molecules of tRNA^HisGUG (the 5'-tRNA^HisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5'-tRNA half molecules and identified the 5'-tRNA^ValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5'-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5'-tRNA^ValCAC/AAC half were highly up-regulated in macrophage EVs …

The Clinical Utility And Diagnostic Implementation Of Human Subject Cell Transdifferentiation Followed By Rna Sequencing, Shenglan Li, Sen Zhao, Jefferson C Sinson, Aleksandar Bajic, Jill A Rosenfeld, Matthew B Neeley, Mezthly Pena, Kim C Worley, Lindsay C Burrage, Monika Weisz-Hubshman, Shamika Ketkar, William J Craigen, Gary D Clark, Seema Lalani, Carlos A Bacino, Keren Machol, Hsiao-Tuan Chao, Lorraine Potocki, Lisa Emrick, Jennifer Sheppard, My T T Nguyen, Anahita Khoramnia, Paula Patricia Hernandez, Sandesh Cs Nagamani, Zhandong Liu, Christine M Eng, Brendan Lee, Pengfei Liu

Faculty, Staff and Students Publications

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary …

Trna Anticodon Cleavage By Target-Activated Crispr-Cas13a Effector, Ishita Jain, Matvey Kolesnik, Konstantin Kuznedelov, Leonid Minakhin, Natalia Morozova, Anna Shiriaeva, Alexandr Kirillov, Sofia Medvedeva, Alexei Livenskyi, Laura Kazieva, Kira S Makarova, Eugene V Koonin, Sergei Borukhov, Konstantin Severinov, Ekaterina Semenova

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus …

Exploration Of Potential Broad-Spectrum Antiviral Targets In The Enterovirus Replication Element: Identification Of Six Distinct 5' Cloverleaves, Morgan G. Daniels, Meagan E. Werner, Rockwell T. Li, Steven M. Pascal

Chemistry & Biochemistry Faculty Publications

Enterovirus genomic replication initiates at a predicted RNA cloverleaf (5′CL) at the 5′ end of the RNA genome. The 5′CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5′CL conservation and divergence for 209 human health-related serotypes from the enterovirus genus, including enterovirus and rhinovirus species. Phylogenetic analysis indicates six distinct 5′CL serotypes that only partially correlate with the species definition. Additional findings include that 5′CL sequence conservation is higher between the EV species than between the RV species, the 5′CL of EVA and EVB are nearly identical, and RVC has the …

Combined Bioinformatic And Splicing Analysis Of Likely Benign Intronic And Synonymous Variants Reveals Evidence For Pathogenicity, Owen R Hirschi, Stephanie A Felker, Surya P Rednam, Kelly L Vallance, D Williams Parsons, Angshumoy Roy, Gregory M Cooper, Sharon E Plon

Faculty, Staff and Students Publications

PURPOSE: Clinical variant analysis pipelines likely have poor sensitivity to the effects on splicing from variants beyond 10 to 20 bases of exon-intron boundaries. Here, we demonstrate the value of SpliceAI to inform curation of rare variants previously classified as benign/likely benign (B/LB) under current guidelines.

METHODS: Exome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored >10 by combined annotation-dependent depletion. Rare synonymous or intronic B/LB variants in 61 genes submitted to ClinVar were …

Current Perspectives On Mass Spectrometry-Based Immunopeptidomics: The Computational Angle To Tumor Antigen Discovery, Bing Zhang, Michal Bassani-Sternberg

Faculty, Staff and Students Publications

Identification of tumor antigens presented by the human leucocyte antigen (HLA) molecules is essential for the design of effective and safe cancer immunotherapies that rely on T cell recognition and killing of tumor cells. Mass spectrometry (MS)-based immunopeptidomics enables high-throughput, direct identification of HLA-bound peptides from a variety of cell lines, tumor tissues, and healthy tissues. It involves immunoaffinity purification of HLA complexes followed by MS profiling of the extracted peptides using data-dependent acquisition, data-independent acquisition, or targeted approaches. By incorporating DNA, RNA, and ribosome sequencing data into immunopeptidomics data analysis, the proteogenomic approach provides a powerful means for identifying …

Neurodevelopmental Deficits And Cell-Type-Specific Transcriptomic Perturbations In A Mouse Model Of Hnrnpu Haploinsufficiency, Sarah A Dugger, Ryan S Dhindsa, Gabriela De Almeida Sampaio, Andrew K Ressler, Elizabeth E Rafikian, Sabrina Petri, Verity A Letts, Jiajie Teoh, Junqiang Ye, Sophie Colombo, Yueqing Peng, Mu Yang, Michael J Boland, Wayne N Frankel, David B Goldstein

Faculty, Staff and Students Publications

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory …

Rna World And The Development Of Rna Protocells, Benjamin C. Mayfield

PANDION: The Osprey Journal of Research and Ideas

Origins of life research, also known as pre-biotic chemistry or astrobiology, aims to unravel the mystery of the first cell’s origin on Earth. This interdisciplinary field encompasses biology, chemistry, and physics, with the primary goal of understanding the conditions necessary for life to emerge from abiotic environments. The RNA world hypothesis suggests that early life initially used RNA instead of DNA to store genomic information and for enzymatic functions. Protocells, membrane-bound entities with metabolic processes and self-replication capabilities, likely preceded the emergence of true cells. The challenges associated with RNA world is currently an active field of research. Advancements in …

Single Cell Multiomics Identifies Cells And Genetic Networks Underlying Alveolar Capillary Dysplasia, Minzhe Guo, Kathryn A Wikenheiser-Brokamp, Joseph A Kitzmiller, Cheng Jiang, Guolun Wang, Allen Wang, Sebastian Preissl, Xiaomeng Hou, Justin Buchanan, Justyna A Karolak, Yifei Miao, David B Frank, William J Zacharias, Xin Sun, Yan Xu, Mingxia Gu, Pawel Stankiewicz, Vladimir V Kalinichenko, Jennifer A Wambach, Jeffrey A Whitsett

Faculty, Staff and Students Publications

Rationale

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown.

Objectives

To identify cell types, gene networks, and cell–cell interactions underlying the pathogenesis of ACDMPV.

Methods

We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs.

Measurements and Main Results

Pathogenic single-nucleotide variants and copy-number …

Genetic Control Of Mrna Splicing As A Potential Mechanism For Incomplete Penetrance Of Rare Coding Variants, Jonah Einson, Dafni Glinos, Eric Boerwinkle, Peter Castaldi, Dawood Darbar, Mariza De Andrade, Patrick Ellinor, Myriam Fornage, Stacey Gabriel, Soren Germer, Richard Gibbs, Craig P Hersh, Jill Johnsen, Robert Kaplan, Barbara A Konkle, Charles Kooperberg, Rami Nassir, Ruth J F Loos, Deborah A Meyers, Braxton D Mitchell, Bruce Psaty, Ramachandran S Vasan, Stephen S Rich, Michael Rienstra, Jerome I Rotter, Aabida Saferali, Moore Benjamin Shoemaker, Edwin Silverman, Albert Vernon Smith, Nhlbi Trans-Omics For Precision Medicine (Topmed) Consortium; Pejman Mohammadi, Pejman Mohammadi, Stephane E Castel, Ivan Iossifov, Tuuli Lappalainen

Faculty, Staff and Student Publications

Exonic variants present some of the strongest links between genotype and phenotype. However, these variants can have significant inter-individual pathogenicity differences, known as variable penetrance. In this study, we propose a model where genetically controlled mRNA splicing modulates the pathogenicity of exonic variants. By first cataloging exonic inclusion from RNA-sequencing data in GTEx V8, we find that pathogenic alleles are depleted on highly included exons. Using a large-scale phased whole genome sequencing data from the TOPMed consortium, we observe that this effect may be driven by common splice-regulatory genetic variants, and that natural selection acts on haplotype configurations that reduce …