Publications by authors named "Matthew D Simon"

49 Publications

Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.

Mol Cell 2021 02 4;81(3):502-513.e4. Epub 2021 Jan 4.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06536, USA; Howard Hughes Medical Institute, Yale University, New Haven, CT 06536, USA. Electronic address:

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.
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http://dx.doi.org/10.1016/j.molcel.2020.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867636PMC
February 2021

Discovery of cellular substrates of human RNA-decapping enzyme DCP2 using a stapled bicyclic peptide inhibitor.

Cell Chem Biol 2020 Dec 18. Epub 2020 Dec 18.

Department of Chemistry, Yale University, New Haven, CT 06520, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06529, USA. Electronic address:

DCP2 is an RNA-decapping enzyme that controls the stability of human RNAs that encode factors functioning in transcription and the immune response. While >1,800 human DCP2 substrates have been identified, compensatory expression changes secondary to genetic ablation of DCP2 have complicated a complete mapping of its regulome. Cell-permeable, selective chemical inhibitors of DCP2 could provide a powerful tool to study DCP2 specificity. Here, we report phage display selection of CP21, a bicyclic peptide ligand to DCP2. CP21 has high affinity and selectivity for DCP2 and inhibits DCP2 decapping activity toward selected RNA substrates in human cells. CP21 increases formation of P-bodies, liquid condensates enriched in intermediates of RNA decay, in a manner that resembles the deletion or mutation of DCP2. We used CP21 to identify 76 previously unreported DCP2 substrates. This work demonstrates that DCP2 inhibition can complement genetic approaches to study RNA decay.
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http://dx.doi.org/10.1016/j.chembiol.2020.12.003DOI Listing
December 2020

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

The NBDY Microprotein Regulates Cellular RNA Decapping.

Biochemistry 2020 10 15;59(42):4131-4142. Epub 2020 Oct 15.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in knockout (KO) cells reveals dysregulated stability of more than 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in KO cells, which correlates with 5' UTR length. deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay factors in KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.
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http://dx.doi.org/10.1021/acs.biochem.0c00672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682656PMC
October 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Global Profiling of Cellular Substrates of Human Dcp2.

Biochemistry 2020 11 14;59(43):4176-4188. Epub 2020 May 14.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Decapping is the first committed step in 5'-to-3' RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remains incompletely mapped, and no consensus sequence or property is currently known to unambiguously predict decapping enzyme substrates. Dcp2 was the first-identified and best-studied eukaryotic decapping enzyme, but it has been shown to regulate the stability of <400 transcripts in mammalian cells to date. Here, we globally profile changes in the stability of the human transcriptome in Dcp2 knockout cells via TimeLapse-seq. We find that P-body enrichment is the strongest correlate of Dcp2-dependent decay and that modification with mA exhibits an additive effect with P-body enrichment for Dcp2 targeting. These results are consistent with a model in which P-bodies represent sites where translationally repressed transcripts are sorted for decay by soluble cytoplasmic decay complexes through additional molecular marks.
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http://dx.doi.org/10.1021/acs.biochem.0c00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641959PMC
November 2020

Author Correction: Enhanced nucleotide chemistry and toehold nanotechnology reveals lncRNA spreading on chromatin.

Nat Struct Mol Biol 2020 Apr;27(4):400

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0413-9DOI Listing
April 2020

Enhanced nucleotide chemistry and toehold nanotechnology reveals lncRNA spreading on chromatin.

Nat Struct Mol Biol 2020 03 10;27(3):297-304. Epub 2020 Mar 10.

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.

Understanding the targeting and spreading patterns of long non-coding RNAs (lncRNAs) on chromatin requires a technique that can detect both high-intensity binding sites and reveal genome-wide changes in spreading patterns with high precision and confidence. Here we determine lncRNA localization using biotinylated locked nucleic acid (LNA)-containing oligonucleotides with toehold architecture capable of hybridizing to target RNA through strand-exchange reaction. During hybridization, a protecting strand competitively displaces contaminating species, leading to highly specific RNA capture of individual RNAs. Analysis of Drosophila roX2 lncRNA using this approach revealed that heat shock, unlike the unfolded protein response, leads to reduced spreading of roX2 on the X chromosome, but surprisingly also to relocalization to sites on autosomes. Our results demonstrate that this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of lncRNAs on chromatin.
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http://dx.doi.org/10.1038/s41594-020-0390-zDOI Listing
March 2020

p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

Mol Cell 2020 02 20;77(4):761-774.e8. Epub 2020 Jan 20.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA. Electronic address:

The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.
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http://dx.doi.org/10.1016/j.molcel.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184554PMC
February 2020

Principles and Practices of Hybridization Capture Experiments to Study Long Noncoding RNAs That Act on Chromatin.

Cold Spring Harb Perspect Biol 2019 11 1;11(11). Epub 2019 Nov 1.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.

The diverse roles of cellular RNAs can be studied by purifying RNAs of interest together with the biomolecules they bind. Biotinylated antisense oligonucleotides that hybridize specifically to the RNA of interest provide a general approach to develop affinity reagents for these experiments. Such oligonucleotides can be used to enrich endogenous RNAs from cross-linked chromatin extracts to study the genomic binding sites of RNAs. These hybridization capture protocols are evolving modular experiments that are compatible with a range of cross-linkers and conditions. This review discusses the principles of these hybridization capture experiments as well as considerations and controls necessary to interpret the resulting data without being misled by artifactual signals.
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http://dx.doi.org/10.1101/cshperspect.a032276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824240PMC
November 2019

Reengineering a tRNA Methyltransferase To Covalently Capture New RNA Substrates.

J Am Chem Soc 2019 11 28;141(44):17460-17465. Epub 2019 Oct 28.

Department of Molecular Biophysics & Biochemistry , Yale University , New Haven , Connecticut 06511 , United States.

Covalent RNA modifications can alter the function and metabolism of RNA transcripts. Altering the RNA substrate specificities of the enzymes that install these modifications can provide powerful tools to study and manipulate RNA. To develop new tools and probe the plasticity of the substrate specificity of one of these enzymes, we examined the engineerability of the uridine-54 tRNA methyltransferase, TrmA. Starting from a mutant that remains covalently bound to its substrate RNA (E358Q, TrmA*), we were able to use both rational design and a high-throughput sequencing assay to examine the RNA substrates of TrmA*. Although rational engineering substantially changed TrmA* specificity, the rationally designed substrate mutants we developed still retained activity with the wild-type protein. Using high-throughput substrate screening of additional TrmA* mutants, we identified a triple mutant of the substrate RNA (C56A;A58G;C60U) that is efficiently trapped by a TrmA* double mutant (E49R;R51E) but not by the wild-type TrmA*. This work establishes a foundation for using protein engineering to reconfigure substrate specificities of RNA-modifying enzymes and covalently trap RNAs with engineered proteins.
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http://dx.doi.org/10.1021/jacs.9b08529DOI Listing
November 2019

Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice.

Cell 2019 04 4;177(3):639-653.e15. Epub 2019 Apr 4.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA; Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, NY 10027, USA; New York Genome Center, New York, NY 10013, USA. Electronic address:

Stochastic activation of clustered Protocadherin (Pcdh) α, β, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.
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http://dx.doi.org/10.1016/j.cell.2019.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823843PMC
April 2019

Stella's Role in Oocyte DNA Methylation Suggests Additional Activities of DNMT1.

Biochemistry 2019 04 28;58(14):1833-1834. Epub 2019 Mar 28.

Department of Molecular Biophysics & Biochemistry , Yale University , New Haven , Connecticut 06511 , United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00146DOI Listing
April 2019

Carbodiimide reagents for the chemical probing of RNA structure in cells.

RNA 2019 01 2;25(1):135-146. Epub 2018 Nov 2.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.

Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide -cyclohexyl--(2-morpholinoethyl)carbodiimide metho--toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3' extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.
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http://dx.doi.org/10.1261/rna.067561.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298570PMC
January 2019

Gaining insight into transcriptome-wide RNA population dynamics through the chemistry of 4-thiouridine.

Wiley Interdiscip Rev RNA 2019 01 28;10(1):e1513. Epub 2018 Oct 28.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.

Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e., altered rate of transcription vs. degradation). The dynamics of different RNA populations can be studied by targeted incorporation of noncanonical nucleosides. 4-Thiouridine (s U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s U metabolic labeling, as well as a discussion of considerations and challenges when designing s U metabolic labeling experiments. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Turnover and Surveillance > Regulation of RNA Stability.
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http://dx.doi.org/10.1002/wrna.1513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768404PMC
January 2019

Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-Thioguanosine.

J Am Chem Soc 2018 11 24;140(44):14567-14570. Epub 2018 Oct 24.

Department of Molecular Biophysics & Biochemistry , Yale University , New Haven , Connecticut 06511 , United States.

RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (sU), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (sG). Using TimeLapse sequencing (TimeLapse-seq), sG can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of sG recoding experiments to reveal transcriptome-wide RNA population dynamics.
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http://dx.doi.org/10.1021/jacs.8b08554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779120PMC
November 2018

Solid phase chemistry to covalently and reversibly capture thiolated RNA.

Nucleic Acids Res 2018 08;46(14):6996-7005

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.

Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.
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http://dx.doi.org/10.1093/nar/gky556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101502PMC
August 2018

TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding.

Nat Methods 2018 03 22;15(3):221-225. Epub 2018 Jan 22.

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, USA.

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.
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http://dx.doi.org/10.1038/nmeth.4582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831505PMC
March 2018

Catching RNAs on chromatin using hybridization capture methods.

Brief Funct Genomics 2018 03;17(2):96-103

The growing appreciation of the importance of long noncoding RNAs (lncRNAs), together with the awareness that some of these RNAs are associated with chromatin, has inspired the development of methods to detect their sites of interaction on a genome-wide scale at high resolution. Hybridization capture methods combine antisense oligonucleotide hybridization with enrichment of RNA from cross-linked chromatin extracts. These techniques have provided insight into lncRNA localization and the interactions of lncRNAs with protein to better understand biological roles of lncRNAs. Here, we review the core principles of hybridization capture methods, focusing on the three most commonly used protocols: capture hybridization analysis of RNA targets (CHART), chromatin isolation by RNA purification (ChIRP) and RNA affinity purification (RAP). We highlight the general principles of these techniques and discuss how differences in experimental procedures present distinct challenges to help researchers using these protocols or, more generally, interpreting the results of hybridization capture experiments.
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http://dx.doi.org/10.1093/bfgp/elx038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888980PMC
March 2018

Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA.

Biochemistry 2017 09 18;56(35):4713-4721. Epub 2017 Aug 18.

Department of Molecular Biophysics & Biochemistry, Yale University , New Haven, Connecticut 06511, United States.

Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given the variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely nonoverlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
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http://dx.doi.org/10.1021/acs.biochem.7b00323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648349PMC
September 2017

mA mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

Nature 2017 08 9;548(7667):338-342. Epub 2017 Aug 9.

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

N-methyladenosine (mA) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that mA influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of mA 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by mA, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that mA has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of mA modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.
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http://dx.doi.org/10.1038/nature23450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729908PMC
August 2017

Enriching s U-RNA Using Methane Thiosulfonate (MTS) Chemistry.

Curr Protoc Chem Biol 2016 Dec 7;8(4):234-250. Epub 2016 Dec 7.

Chemical Biology Institute, Yale University, West Haven, Connecticut.

Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s U-RNA is the efficiency of the chemistry used to enrich s U-RNA. Here, we present a protocol for s U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s U to study RNA biology. © 2016 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpch.12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161349PMC
December 2016

Capture Hybridization Analysis of DNA Targets.

Methods Mol Biol 2016 ;1480:87-97

Department of Molecular Biophysics & Biochemistry, Yale University, 27392, New Haven, CT, 06511, USA.

There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells.
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http://dx.doi.org/10.1007/978-1-4939-6380-5_8DOI Listing
January 2018

The Properties of Long Noncoding RNAs That Regulate Chromatin.

Annu Rev Genomics Hum Genet 2016 08 21;17:69-94. Epub 2016 Apr 21.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511; email: , ,

Beyond coding for proteins, RNA molecules have well-established functions in the posttranscriptional regulation of gene expression. Less clear are the upstream roles of RNA in regulating transcription and chromatin-based processes in the nucleus. RNA is transcribed in the nucleus, so it is logical that RNA could play diverse and broad roles that would impact human physiology. Indeed, this idea is supported by well-established examples of noncoding RNAs that affect chromatin structure and function. There has been dramatic growth in studies focused on the nuclear roles of long noncoding RNAs (lncRNAs). Although little is known about the biochemical mechanisms of these lncRNAs, there is a developing consensus regarding the challenges of defining lncRNA function and mechanism. In this review, we examine the definition, discovery, functions, and mechanisms of lncRNAs. We emphasize areas where challenges remain and where consensus among laboratories has underscored the exciting ways in which human lncRNAs may affect chromatin biology.
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http://dx.doi.org/10.1146/annurev-genom-090314-024939DOI Listing
August 2016

Probing Xist RNA Structure in Cells Using Targeted Structure-Seq.

PLoS Genet 2015 Dec 8;11(12):e1005668. Epub 2015 Dec 8.

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, United States of America.

The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function.
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http://dx.doi.org/10.1371/journal.pgen.1005668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672913PMC
December 2015

Insight into lncRNA biology using hybridization capture analyses.

Authors:
Matthew D Simon

Biochim Biophys Acta 2016 Jan 14;1859(1):121-7. Epub 2015 Sep 14.

Dept. of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06516, USA; Chemical Biology Institute, Yale West Campus, West Haven, CT, 06511, USA. Electronic address:

Despite mounting evidence of the importance of large non-coding RNAs (lncRNAs) in biological regulation, we still know little about how these lncRNAs function. One approach to understand the function of lncRNAs is to biochemically purify endogenous lncRNAs from fixed cells using complementary oligonucleotides. These hybridization capture approaches can reveal the genomic localization of lncRNAs, as well as the proteins and RNAs with which they interact. To help researchers understand how these tools can uncover lncRNA function, this review discusses the considerations and influences of different parameters, (e.g., crosslinking reagents, oligonucleotide chemistry and hybridization conditions) and controls to avoid artifacts. By examining the application of these tools, this review will highlight the progress and pitfalls of studying lncRNAs using hybridization capture approaches.This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.
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http://dx.doi.org/10.1016/j.bbagrm.2015.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707088PMC
January 2016

Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.

Mol Cell 2015 Sep;59(5):858-66

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA. Electronic address:

We describe a chemical method to label and purify 4-thiouridine (s(4)U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s(4)U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s(4)U labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA, such as 4-thiouridine tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.
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http://dx.doi.org/10.1016/j.molcel.2015.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560836PMC
September 2015

The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites.

Mol Cell 2014 Sep 21;55(5):791-802. Epub 2014 Aug 21.

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Mechanistic roles for many lncRNAs are poorly understood, in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin, and that localization responds to cues involved in the transcription process.
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http://dx.doi.org/10.1016/j.molcel.2014.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428586PMC
September 2014

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Cell 2014 May;157(4):869-81

Division of Newborn Medicine, Boston Children's Hospital, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.
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http://dx.doi.org/10.1016/j.cell.2014.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038154PMC
May 2014

High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation.

Nature 2013 Dec 27;504(7480):465-469. Epub 2013 Oct 27.

Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02114.

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X chromosome (Xi) and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre (Xic). How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.
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http://dx.doi.org/10.1038/nature12719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904790PMC
December 2013