Talk abstracts
Friday 01:15-01:30pm: In vivo RNA structural probing of uracil and guanine base pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
David Mitchell III (Department of Chemistry, Penn State University), Andrew J. Renda (Department of Biochemistry and Molecular Biology, Penn State University), Catherine A. Douds (Department of Chemistry, Penn State University), Paul Babitzke (Department of Biochemistry and Molecular Biology, Penn State University), Sarah M. Assmann (Department of Biology, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Penn State University)
Abstract:
Many biological functions performed by RNAs arise from their in vivo structures. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base pairing and so are of value to determining structures adopted by RNAs. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules, ranging from protons to secondary metabolites to proteins. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor ~1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base pairing status of all four RNA bases.
References:
Bevilacqua PC, Assmann SM. 2018. Technique development for probing RNA structure in vivo and genome-wide. In Additional Perspectives on RNA Worlds, (ed. TR Cech, JA Steitz, JF Atkins). Cold Spring Harbor Laboratory Press, New York, NY (in press).
Mitchell D, 3rd, Ritchey LE, Park H, Babitzke P, Assmann SM, Bevilacqua PC. 2018. Glyoxals as in vivo RNA structural probes of guanine base-pairing. RNA 24: 114-124.
Ritchey LE, Su Z, Tang Y, Tack DC, Assmann SM, Bevilacqua PC. 2017. Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo. Nucleic Acids Res.
Rouskin S, Zubradt M, Washietl S, Kellis M, Weissman JS. 2014. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505: 701-705.
Keywords: in vivo RNA probing, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, RNA structure
Friday 01:30-01:45pm: Maximizing quantitative structural information from high-throughput RNA structure probing
Molly E. Evans (Department of Chemical and Biological Engineering, Northwestern University), Angela M Yu (Tri-institutional Training Program in Computational Biology and Medicine, Weill Cornell Medicine), Petr Sulc (Biodesign Center for Molecular Design and Biomimetics, Arizona State University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)
Abstract:
RNAs enact numerous cellular functions through their formation of intricately folded structures. High-throughput RNA structure probing experiments that couple chemical probing of RNA structure to high-throughput sequencing can be used to determine signatures of these biologically relevant structures that can be used to construct models of functional RNA folds. While this experimental approach has so far yielded useful data, several major limitations have precluded our ability to obtain precise and quantitative RNA structural information, including a lack of standards for experimental and data processing steps that result in inconsistent generation and interpretation of the primary chemical probing ‘reactivity’ data that is generated by these experiments. This in turn has prevented rigorous comparison of experimental results within and between experiments.
Here, we have designed and begun to characterize a standard benchmark panel of RNAs of known structure that can be used as experimental calibration standards to allow comparison of reactivities within and between experiments. By implementing calibration standards, measurements of reactivity in RNA structure probing experiments will become more quantitative, allowing the maximal amount of structural information to be extracted from these experiments across laboratories. Additionally, in order to precisely define measured reactivity, we have leveraged our expertise in RNA design by creating a panel of RNAs that vary in sequence but are designed to fold into the same simple secondary structure. Our preliminary studies show that using improved experimental methods, chemical probing reactivities match to the predicted secondary structures of the first panel of RNAs. These RNAs will be used as a starting point to develop and validate an accurate and quantitative definition of chemical probe reactivity that is directly linked to RNA structure.
Keywords: high-throughput structure probing, standardization, SHAPE-Seq
Friday 01:45-02:00pm: Title not available online - please see the printed booklet.
Jane K Frandsen (Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University), Anna V. Sherwood (Molecular Cellular and Developmental Biology Graduate Program, Center for RNA Biology, The Ohio State University), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology Graduate Program, Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 02:00-02:15pm: KIN-CLIP: transcriptome-wide kinetics for RNA-protein interactions in cells
Deepak Sharma (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106), Leah Zagore (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106), Matthew Brister (Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106), Carlos Crespo Hernandez (Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106), Donny Licatalosi (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106), Eckhard Jankowsky (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106)
Abstract:
RNA-binding proteins (RBPs) often interact with many different RNAs at sometimes large numbers of binding sites. These interactions are highly dynamic. Association and dissociation rate constants by which an RBP interacts with each binding site thus determine global RNA binding patterns of the protein and ultimately its biological function. However, it has not been possible to measure kinetic parameters for protein binding at individual RNA sites in cells.
Here, we describe a transcriptome-wide approach to determine kinetic parameters for protein binding at individual RNA sites in cells. We combine time-resolved UV-crosslinking with a pulsed UV-laser, Immunoprecipitation, Next Generation Sequencing, and large scale kinetic modeling, to determine rate constants for association, dissociation, crosslinking as well as the fractional occupancy for thousands of binding sites for the mouse RBP Dazl in GC1 cells. This kinetic CLIP (KIN-CLIP) approach reveals that both association and dissociation rate constants for Dazl vary by 2 to 3 orders of magnitude among different binding sites, thus providing Dazl with a large dynamic range for binding to various sites. Crosslinking rate constants differ by only about one order of magnitude over all binding sites. Our data reveal that Dazl stays bound at its binding sites for only few seconds or less, and that discrimination between different binding sites occurs predominantly during the association step. The fractional occupancy for a majority of Dazl binding sites is smaller than ten percent, suggesting that regulation of binding site accessibility plays a large role for Dazl-RNA binding in the cell. We finally use multivariate machine learning to correlate the kinetic parameters with RNA features, cellular pathways, and ribosome profiling data and devise a detailed, quantitative model that links Dazl’s RNA binding kinetics to its impact on mRNA metabolism.
Collectively, our results show that the KIN-CLIP technique, by bridging biochemical and transcriptomic approaches, allows the measurement of previously inaccessible quantitative and biochemical parameters for RNA-protein interactions in cells and the use of these datasets can aid in generating quantitative mechanistic models of RNA-protein interactions in vivo.
Keywords: in vivo biochemistry, RNA binding kinetics, Multivariate RNA regulation
Friday 02:15-02:30pm: Title not available online - please see the printed booklet.
Audrey C. Kehling (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Mi Seul Park (Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.), Alison E. Hager (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.), Kotaro Nakanishi (Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.)
Abstract not available online - please check the printed booklet.
Friday 02:30-02:45pm: Title not available online - please see the printed booklet.
Hiba A. Al-Ashtal (University of Pittsburgh), Courtney M. Rubottom (University of Pittsburgh), Andrea J. Berman (University of Pittsburgh)
Abstract not available online - please check the printed booklet.
Friday 03:15-03:30pm: Accurate transcriptome-wide identification of m5C RNA modification events at single molecule resolution from direct RNA sequencing of human cell lines
Raja Shekar Varma Kadumuri (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Mir Quoseena (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University.)
Abstract:
N5-methyl Cytosine (m5C) is an abundant chemical modification of RNA known to regulate RNA processing[1], stability[2], mRNA export[3], cleavage and translation processes. Although recent methods have enabled the epitranscriptome profiling of m5C in various cell lines and tissues[3], current sequencing based methods have some major drawbacks including multifaceted labor-intensive protocols, cross-reactivity to chemical compounds and ambiguous mapping of m5C positions resulting from short read sequencing technologies[4]. Recent nanopore-based direct RNA sequencing provides an opportunity to simultaneously obtain full length isoforms and m5C modification detection with reduced labor-intensive processes and better accuracy[5, 6]. However, currently there is no tool to detect RNA modifications using Direct RNA sequencing data. Here, we present RAVEN, a deep neural network-based framework developed mainly on Convolutional Neural Network (CNN) architecture, for the detection of m5C modifications from nanopore-based direct RNA sequencing data. Raven is trained and validated on ~10,000 experimentally known N5-methyl Cytosine (m5C) and unmodified Cytosine (C) signal signatures[3] from matched polyA direct RNA sequencing and bisulfite treated short read sequencing data, generated in a human Hela cell line. Raven takes the albacore base called fast5 files resulting from direct RNA sequencing as input to provide m5C modification predictions at read level with an accuracy ~80%, along with a statistical probability of an m5C modification at a genomic loci considering the depth of the aligned reads. Raven also enables machine learning models including K-Nearest neighbor and Ada boost classifiers which were found to show 73-75% accuracy for m5C prediction. Current version of Raven can be accessed at (github wiki page: https://github.iu.edu/rkadumu/Raven/wiki)
References:
1 Kadumuri, R.V. and Janga, S.C. (2018) Epitranscriptomic Code and Its Alterations in Human Disease. Trends Mol Med
2 Hussain, S., et al. (2013) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome biology 14, 215
3 Yang, X., et al. (2017) 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell research 27, 606-625
4 Wreczycka, K., et al. (2017) Strategies for analyzing bisulfite sequencing data. Journal of biotechnology 261, 105-115
5 Qian Liu, D.C.G., Dieter Egli, Kai Wang (2018) NanoMod: a computational tool to detect DNA modifications using Nanopore long-read sequencing data. bioRxiv
6 Marcus H Stoiber, J.Q., Rob Egan, Ji Eun Lee, Susan E Celniker, Robert Neely, Nicholas Loman, Len Pennacchio, James B Brown (2017) De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing. bioRxiv
Keywords: N5-methyl cytosine modification, Deep learning neural network, Direct RNA nanopore sequencing
Friday 03:30-03:45pm: DNA methylation regulates mRNA alternative cleavage and polyadenylation
Vishal Nanavaty (Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic), Changjing Hong (Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic), Elizabeth Abrash, Jeffrey M. Bhasin, Thomas J. Sweet (Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic), Tae Hyun Hwang (Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic), Angela H. Ting (Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic)
Abstract not available online - please check the printed booklet.
Friday 03:45-04:00pm: The association of tRNA intron accumulation with environmental stress conditions
Lauren Peltier (Molecular Genetics, The Ohio State University), Alicia Bao (Molecular Genetics, The Ohio State University), Anita Hopper (Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 04:00-04:15pm: An orphan 3’-5’ polymerase implicated in noncoding RNA processing
Samantha Dodbele (Ohio State Biochemistry Program, The Ohio State University OSU; Center for RNA Biology, OSU), Blythe Moreland (Center for RNA Biology, OSU; Department of Physics, OSU), Yicheng Long (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU), Ralf Bundschuh (Center for RNA Biology, OSU; Department of Physics, OSU; Division of Hematology and Department of Internal Medicine, OSU), Jane Jackman (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU; Department of Chemistry and Biochemistry, OSU)
Abstract not available online - please check the printed booklet.
Friday 04:15-04:30pm: Maturation of Drosophila intronic-RNase P RNA (RPR) critically depends on association with its protein subunits
Geeta Palsule (Department of Molecular Genetics, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Amanda Simcox (Department of Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 08:45-09:00am: Final maturation stages of the polypeptide exit tunnel
Daniel Wilson (Biological Sciences Carnegie Mellon University), Amber LaPeruta (Biological Sciences Carnegie Mellon University), John L. Woolford (Biological Sciences Carnegie Mellon University)
Abstract not available online - please check the printed booklet.
Saturday 09:00-09:15am: Quantitative analysis of yeast Kozak variants for AUG and near-AUG start codons using a massively parallel reporter assay
Christina Akirtava (Biological Sciences at Carnegie Mellon University), Hunter Kready (Biological Sciences at Carnegie Mellon University), Lauren Nazzaro (Biological Sciences at Carnegie Mellon University), Gemma E. May (Biological Sciences at Carnegie Mellon University), C. Joel McManus (Biological Sciences at Carnegie Mellon University)
Abstract not available online - please check the printed booklet.
Saturday 09:15-09:30am: Discovery and characterization of peptide inhibitors of RsmC function
Davidnhan To (Department of Chemistry and Biochemistry, Kent State University), Keshav GC (Department of Chemistry and Biochemistry, Kent State University), Dr. Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)
Abstract not available online - please check the printed booklet.
Saturday 09:30-09:45am: Rusty ribosomes: how oxidation damages rRNA and affects translation
Jessica Willi (Department of Chemistry and Biochemistry, University of Bern), Norbert Polacek (Department of Chemistry and Biochemistry, University of Bern), Pat A. Limbach (Department of Chemistry, University of Cincinnati)
Abstract:
Oxidative stress is emerging as a serious source of damage to RNA, which might contribute to numerous aspects of aging and diseases such as Alzheimer’s, Parkinson’s, Diabetes, and Huntington’s. Among all RNAs, the ribosomal RNA (rRNA) stands out for its central role in catalyzing protein synthesis.
We found that oxidative stress indeed damages rRNA and disrupts in vitro translation capabilities in a dose-dependent manner [1]. Using an established chemical engineering approach [2], we have introduced single base oxidations 8-oxorA, 8-oxorG, 5-HOrC and 5-HOrU into the catalytic heart of the ribosome, the peptidyl transferase center. This revealed how oxidation can influence ribosomal function: strength and type of the effect heavily depends on the position of the base oxidized, ranging from no effect to almost complete inhibition of translation [1].
Additionally, we employ immunoprecipitation sequencing (IP-Seq) and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3] to quantify and map where rRNA oxidations occur both in vitro and in vivo. These oxidation maps enable us to link previous biochemical assays to biology and deepen our molecular understanding of the ribosomal machinery as well as how translation fails in age-related diseases.
References:
[1] Willi, J., Küpfer, P., Evéquoz, D., Fernandez, G., Katz, A., Leumann, C. & Polacek, N. Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center. Nucleic Acids Res. 46, 1945–1957 (2018).
[2] Erlacher, M. D., Chirkova, A., Voegele, P. & Polacek, N. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis. Nat. Protoc. 6, 580–92 (2011).
[3] Limbach, P. A. & Paulines, M. J. Going global: the new era of mapping modifications in RNA. Wiley Interdiscip. Rev. RNA (2016).
Keywords: ribosome, oxidative stress, rRNA
Saturday 09:45-10:00am: Pseudouridylation of mRNA can alter translation elongation, fidelity and termination
Monika Franco (Program in Chemical Biology, University of Michigan), Daniel Eyler (Department of Chemistry, University of Michigan), Kristin Koutmou (Department of Chemistry, Program in Chemical Biology, University of Michigan)
Abstract not available online - please check the printed booklet.
Saturday 10:00-10:15am: Ribosome binding by yeast eIF4B drives stress-induced changes in translation
Xiaozhuo LIU (Department of Biological Sciences, The State University of New York at Buffalo), Houtan Moshiri (Department of Biological Sciences, The State University of New York at Buffalo), Sarah E. Walker (Department of Biological Sciences, The State University of New York at Buffalo)
Abstract not available online - please check the printed booklet.
Saturday 10:15-10:30am: MiR-574-FAM210A axis maintains cardiac mitochondrial translational homeostasis
Jiangbin Wu (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY 14586), Kadiam C Venkata Subbaiah (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY 14586), Feng Jiang, (Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, NY 14586), Omar Hedaya (Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, NY 14586), Wai Hong Wilson Tang (Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195), Peng Yao (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY 14586)
Abstract:
Prior studies have examined mechanisms that coordinate mRNA transcription of nuclear-encoded mitochondrial genes (NEMGs) and mitochondrial encoded genes (MEGs). However, regulatory mechanisms of MEG mRNA translation and its coupling with NEMG mRNA translation under disease conditions remain underexplored. Here, we have identified a microRNA-574-FAM210A (family with sequence similarity 210 member A) axis that maintains the optimal translation of MEGs and mitochondrial homeostasis in cardiac cells, as a compensatory cardioprotective pathway to prevent heart failure progression. miR-574 knockout mice exhibited an advanced cardiac hypertrophy phenotype associated with increased fibrosis after cardiac stresses. miR-574 produces two functional strands, miR-574-5p and miR-574-3p, which play synergistic cardioprotective role in preventing ventricular pathological remodeling, partially by downregulating a common target mRNA FAM210A. miR-574-5p antagonizes FAM210A expression in cardiac myocytes to prevent excessive MEG expression, enhanced reactive oxygen species production and impaired mitochondrial activity, thereby preventing myocyte hypertrophy. Hypertrophic stress activates exosome-mediated release of miR-574-3p captured by phospho-hnRNP L (heterogeneous nuclear ribonucleoprotein L) and reduces cardiac fibroblast proliferation by targeting FAM210A. Our results also define a critical role of FAM210A in translational control of a specific cohort of cardiac mitochondrial encoded genes. We found that FAM210A, induced in dilated cardiomyopathy and ischemic heart failure patients, is a mitochondrial translational activator within a complex with EF-Tu and ATAD3A to accelerate translation elongation of specific MEGs. Cardiomyocyte-specific knockout of Fam210a in mice leads to mitochondrial dysfunction, myofiber disarray, and spontaneous cardiac hypertrophy. Collectively, we discover a novel miR-574-FAM210A pathway that modulates cardiac mitochondrial translational homeostasis for maintaining cardiac well-being.
Keywords: translational control, miRNA, Fam210a
Saturday 11:00-11:15am: MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs
Qinyu Sun, Vidisha Tripathi, Deepak Singh, Qinyu Hao, Supriya G. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Je-Hyun Yoon, Kyung-Won Min, Sylvia Davila, Richard W. Zealy, (Department of Biochemistry and Molecular Biology, Medical University of South Carolina), Xiao Ling Li, Ashish Lal (Regulatory RNAs and Cancer Section, Genetics Branch, Center for Cancer Research, National Cancer Institute), Elin Lehrmann, Yongqing Zhang, Kevin G. Becker, Myriam Gorospe (Laboratory of Genetics and Genomics, National Institute of Aging-Intramural Research program, NIH), Susan M. Freier (Ionis Pharmaceuticals Inc, Carlsbad, CA), Kannanganattu Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign)
Abstract:
Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.
Keywords: long non-coding RNA, microRNA host gene , RNA-binding proteins
Saturday 11:15-11:30am: Dynamic regulation of alternative splicing of neurofibromatosis type 1 exon 23a in the mouse brain
Xuan Guo (Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, 44106), Hua Lou (Department of Genetics and Genome Sciences, Center for RNA Molecular Biology, School of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, 44106)
Abstract:
The mechanisms by which alternative splicing is regulated in response to environmental signals are poorly understood. Our group uses neurofibromatosis type 1 (Nf1) as a model to study signaling-induced splicing regulation.
Nf1 is a tumor suppressor gene that contains an alternatively spliced exon, 23a. Our previous studies demonstrate that inclusion of this exon significantly increases Ras/ERK signaling 1,2. As ERK activity changes dynamically during learning, we hypothesize that alternative splicing of Nf1 exon 23a also undergoes dynamic regulation and plays an important role in learning and memory.
To test this hypothesis, we asked if and how inclusion of Nf1 exon 23a changes during learning in wild type mice. To examine different regions of the brain simultaneously, we carried out BaseScope RNA in situ hybridization (ISH) assay, which uses exon junction probes to analyze the splicing pattern in brain slices3. We found that during a fear conditioning learning paradigm, in several regions of the amygdala, inclusion of Nf1 23a displays a dynamic change, increasing five minutes after the mice were shocked and returning to baseline at 24 hours. No significant changes were observed in cortex, hippocampus or other amygdala regions. Interestingly, a concomitant dynamic phospho-ERK level change in amygdala was also observed.
Ketamine is used to treat major depression and has been documented to lead to increased phospho-ERK levels. We carried out similar RNA and protein assays to analyze mouse brain slices prepared from control and treated mice. A similar correlation between increased Nf1 exon 23a inclusion and phospho-ERK levels in ketamine-treated mice was observed.
These data demonstrate that dynamic changes of the alternative splicing pattern of Nf1 exon 23a occur under physiological conditions in vivo, which is correlated with the Ras-ERK activity. We are currently studying the underlying mechanisms of this regulated splicing event.
References:
1. Nguyen H T, Hinman M N, Guo X, et al. Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice[J]. Hum Mol Genet. 2017, 26(19): 3797-3807.
2. Hinman M N, Sharma A, Luo G, et al. Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons[J]. Mol Cell Biol. 2014, 34(12): 2188-2197.
3. Guo X, Zhao Y, Nguyen H, et al. Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay[J]. J Vis Exp. 2018(138).
Keywords: Alternative Splicing, Nf1 exon 23a, Mouse Brain
Saturday 11:30-11:45am: LUC7L2 is a splicing regulatory protein that is frequently mutated in bone marrow neoplasms
Hershberger C.E. (Cleveland Clinic Foundation, Cleveland, OH), Gu, X., Dietrich, R.C., Hiznay J. (Cleveland Clinic Foundation, Cleveland, OH), Hosono N. (University of Fukui, Fukui, Japan), Makishima, H. (Kyoto University, Kyoto, Japan), Saunthararajah, Y., Maciejewski J. (Cleveland Clinic Foundation, Cleveland, OH), Padgett R.A. (Cleveland Clinic Foundation, Cleveland, OH)
Abstract:
Myelodysplastic syndrome (MDS) is a disease distinguished by the large percentage of patients that harbor a mutation in one of several splicing factors. Exome sequencing of patient bone marrow samples recently identified frameshift mutations and deletions encompassing the putative splicing factor gene LUC7L2 in 14% of MDS patients. Low expression of LUC7L2 is correlated with worse prognosis suggesting that LUC7L2-deficiency contributes to the pathology of MDS. While it is known that the essential orthologous yeast protein Luc7p is involved in 5’ splice site recognition, little is understood about the function of mammalian LUC7L2. To understand the pathology of LUC7L2-deficiency, we characterized its role as a splicing factor using co-immunoprecipitation mass spectrometry, CLIP-Seq, RNA-Seq and intron splicing efficiency assays.
In this work, we demonstrate that LUC7L2 interacts with both core and regulatory spliceosomal proteins. LUC7L2 binds selectively to over 300 pre-mRNA transcripts and early acting snRNAs. LUC7L2 binding on pre-mRNA is enriched 20-fold in splice site proximal regions. Knocking down LUC7L2 in HEK293 and K562 cells lines results in changes to constitutive and alternative splicing with removal of alternatively-spliced introns being the group most frequently affected. Validation of target-transcripts confirms increases in intron splicing efficiency, suggesting that LUC7L2 plays the role of a splicing repressor. Parallel analysis of published RNA-Seq datasets for SF3B1, U2AF1 and SRSF2 mutations in K562 cells has revealed commonly mis-spliced targets PRC1, MRPL33, PTBP1 and U2AF1. These transcripts are candidates for further study of the influence of alternatively spliced transcripts on the progression of disease. To our knowledge this is the first description of LUC7L2 as a splicing regulatory factor. This work provides insights into the function of LUC7L2, providing the framework for our intensive examination of its role in MDS in the future.
Keywords: Alternative Splicing, MDS, LUC7L2
Saturday 11:45-12:00pm: Overexpression of a non-muscle RBFOX2 splice isoform induces cardiac arrhythmias in Myotonic Dystrophy
Chaitali Misra (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL), Sushant Bangru, Darren J. Parker (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL), Sara Koenig, Ellen Lubbers, Peter Mohler (Davis Heart and Lung Research Institute, Wexner Medical Center, Ohio State University, OH), Jamila Hedhli, Wawrzyniec L. Dobrucki (Department of Bioengineering, University of Illinois, Urbana-Champaign, IL), Thomas A. Cooper (Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX), Auinash Kalsotra (Department of Biochemistry, Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign)
Abstract not available online - please check the printed booklet.
Saturday 12:00-12:15pm: DDX3X Modulates CGG-Associated RAN Translation and Neuronal Toxicity
Alexander E. Linsalata (University of Michigan), Fang He (Texas A&M University-Kingsville), Sam Natla (University of Michigan), Peter K. Todd (University of Michigan)
Abstract:
Repeat-associated, non-AUG (RAN) translation is a non-canonical mode of protein synthesis distinguished by its dependence on nucleotide-repeat expansions (NREs) and use of non-AUG start codons. It is associated with several neurodegenerative disorders, including Fragile X-associated tremor/ataxia syndrome (FXTAS). The mechanism of RAN translation remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in a Drosophila melanogaster model of FXTAS. We identified the DEAD-box RNA helicase belle/DDX3X as a modifier of both CGG repeat-induced toxicity and RAN translation. Disrupting belle/DDX3X selectively inhibited RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. In addition, in-vitro translation experiments indicated that belle/DDX3X exerts this role co-translationally, rather than at an upstream step in gene expression. These findings implicate RNA secondary structure as a critical element mediating RAN translation and a potential target for treating repeat-associated neurodegeneration.
Keywords: RAN Translation, DDX3X, Neurodegeneration
Saturday 12:15-12:35pm: High levels of intron-containing transcripts in aggressive cancers
Daniel Dominguez (Pharmacology, UNC Chapel Hill), Yi-Tsuan Tsai (Bioinformatics and Computational Biology, UNC Chapel Hill), Zefeng Wang (Chinese Academy of Sciences), Christopher Burge (Biology, MIT)
Abstract:
RNA processing defects can initiate transformation, promote tumor growth, enhance metastasis and confer drug resistance. We performed a comprehensive analysis of hundreds of kidney cancer transcriptomes and uncovered an unusual subset of tumors with abnormally high levels of most detected introns. We termed this subset of tumors, accounting for about 20% of kidney cancer cases, the IR-class (Intron Retention-class). We find that high intron retention levels are linked to a defect in the non-sense mediated decay pathway (NMD), as tumors with high intron retention also showed increased levels of NMD-associated exons (poison cassette exons) and other NMD-targeted transcripts. Analysis of ribosome profiling data revealed some intron-containing species associate with ribosomes, indicative of their presence in the cytoplasm and evasion of NMD. Thus, high levels of intron-containing transcripts are likely a result of defects in NMD rather than defects in splicing. Assessment of thousands of other tumor samples spanning a range of tumor types, revealed that this phenomenon is unique to kidney cancer. While no individual genetic alternation was enriched in the IR-class, IR-class tumors showed recurrent mutations in MTOR pathway members and altered expression of core energy metabolism pathways. Patients with IR-class tumors had a striking decrease in overall survival, living about half as long as patients with non-IR tumors. Analysis of a panel of kidney cancer cell lines revealed a similar pattern, wherein only certain cell lines exhibited high levels of intron retention and NMD-exons, providing tractable models for study of IR-class tumors. Our work links key cancer signaling pathways to NMD defects resulting in high levels of aberrant transcripts in patients with poor clinical outcomes.
Keywords: Intron Retention, Cancer, RNA processing
Saturday 12:35-12:55pm: Regulation of human alternative splicing by the spliceosomal small nuclear RNAs
Heidi Dvinge (UW-Madison), Robert Bradley (Fred Hutchinson Cancer Research Center)
Abstract:
Alternative splicing of pre-mRNAs plays a pivotal role during the establishment and maintenance of human cell types. Characterizing the trans-acting regulatory proteins that control alternative splicing in both healthy and malignant cells has therefore been the focus of much research. Recent work has established that even core protein components of the spliceosome, which are required for splicing to proceed, can nonetheless contribute to splicing regulation by modulating splice site choice. We here demonstrate that the RNA components of the spliceosome likewise influence alternative splicing decisions and contribute to the establishment of global splicing programs. Although these small nuclear RNAs (snRNAs), termed U1, U2, U4, U5, and U6 snRNA, are present in equal stoichiometry within the spliceosome, we found that their relative levels vary by an order of magnitude across tissues, and between normal and malignant cells. Physiologically relevant perturbation of individual snRNAs drove widespread gene-specific differences in alternative splicing, but not transcriptome-wide splicing failure. Our data indicate that endogenous variation in snRNA levels confers regulatory capacity on these RNAs. snRNAs thereby play an unexpectedly complex role in establishing global splicing programs, in addition to their well-characterized roles in basal splicing catalysis. Genes that were particularly sensitive to variations in snRNA abundance in a breast cancer cell line model were likewise preferentially mis-spliced within a clinically diverse cohort of invasive breast ductal carcinomas. As aberrant mRNA splicing is prevalent in many solid and liquid tumors, we propose that a full understanding of dysregulated pre-mRNA processing in cancers requires study of the RNA as well as protein components of the splicing machinery.
Keywords: snRNA, splicing, regulation