DOCSLIB.ORG

Transcriptome-Wide Profiling of Multiple RNA Modifications Simultaneously at Single-Base Resolution,” by Vahid Khoddami, Archana Yerra, Timothy L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transcriptome-Wide Profiling of Multiple RNA Modifications Simultaneously at Single-Base Resolution,” by Vahid Khoddami, Archana Yerra, Timothy L Correction BIOCHEMISTRY, CHEMISTRY Correction for “Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution,” by Vahid Khoddami, Archana Yerra, Timothy L. Mosbruger, Aaron M. Fleming, Cynthia J. Burrows, and Bradley R. Cairns, which was first published March 14, 2019; 10.1073/pnas.1817334116 (Proc Natl Acad Sci USA 116:6784–6789). The authors note that the following statement should be added to the Acknowledgments: “This work was also supported by Grant R01 GM093099 from the NIH/National Institute of General Medical Sciences (to C.J.B.).” Published under the PNAS license. Published online April 22, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1905628116 9136 | PNAS | April 30, 2019 | vol. 116 | no. 18 www.pnas.org Downloaded by guest on October 2, 2021 Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution Vahid Khoddamia,b,c,1,2, Archana Yerrab,c,1, Timothy L. Mosbrugerd, Aaron M. Fleminge, Cynthia J. Burrowse,3, and Bradley R. Cairnsb,c,3 aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; bHoward Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT 84112; cDepartment of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112; dBioinformatics Shared Resource, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112; and eDepartment of Chemistry, University of Utah, Salt Lake City, UT 84112 Contributed by Cynthia J. Burrows, January 25, 2019 (sent for review October 9, 2018; reviewed by Juan D. Alfonzo, Thomas Carell, and Peter C. Dedon) The breadth and importance of RNA modifications are growing First, we provide the conceptual basis for sequencing/mismatch- rapidly as modified ribonucleotides can impact the sequence, struc- based detection of all three modifications (Fig. 1A) and an ex- ture, function, stability, and fate of RNAs and their interactions with ample tRNA (glycine) that illustrates modification clarity within other molecules. Therefore, knowing cellular RNA modifications at our HeLa cell dataset (Fig. 1 B and C, with multiple additional single-base resolution could provide important information regard- examples in SI Appendix, Figs. S1–S3). ing cell status and fate. A current major limitation is the lack of Detection of m5C in RNA (and DNA) relies on differential methods that allow the reproducible profiling of multiple modifi- sensitivity to bisulfite: unmethylated cytosine is efficiently de- cations simultaneously, transcriptome-wide and at single-base res- aminated by bisulfite ions converting cytosine to uridine, which is olution. Here we developed RBS-Seq, a modification of RNA bisulfite subsequently read as thymidine following desulfonation, RT- sequencing that enables the sensitive and simultaneous detection of PCR, and sequencing. In contrast, m5C resists bisulfite and re- m5C, Ψ,andm1A at single-base resolution transcriptome-wide. With A 5 1 mains cytosine after sequencing (15, 21) (Fig. 1 ). We improved RBS-Seq, m C and m A are accurately detected based on known prior m5C profiling methods by combining heat and the strong signature base mismatches and are detected here simultaneously chemical denaturant formamide, which improves RNA dena- along with Ψ sites that show a 1–2 base deletion. Structural anal- turation and bisulfite treatment (which preferentially modifies yses revealed the mechanism underlying the deletion signature, single-stranded RNA), providing a global C → T conversion which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base- Significance skipping during cDNA synthesis. Detection of each of these modifi- cations through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling The field of RNA modification would be significantly advanced covariation studies. Application of RBS-Seq on HeLa RNA revealed by the development of sensitive, accurate, single-base resolu- almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and tion methods for profiling multiple common RNA modifications provided hundreds of new m5CandΨ sites in noncoding RNAs and in the same RNA molecule. Our work provides several advances mRNAs. However, our results diverge greatly from earlier work, toward that goal, including (i) quantitative methods for pro- suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs filing Ψ sites at true base-pair resolution transcriptome-wide, and the absence of substantial m1A in mRNAs. Taken together, the (ii) a chemical understanding of our observed Ψ-dependent 5 approaches and refined datasets in this work will greatly enable deletion signature, (iii) improved methods for profiling m C 1 future epitranscriptome studies. and m A, and (iv) a coupling of these methods for the simul- taneous detection of all three modifications in the same RNA. Together, the combinatorial ability and relative ease of exe- RNA modification | pseudouridine | RNA methylation | m1A | methyl cution provided by this procedure should greatly forward adenosine epitranscriptome studies involving these three very common RNA modifications. Covalent modifications of RNA are numerous (1), and transcriptome-wide profiling enables broad and systematic Author contributions: V.K. and B.R.C. designed research; V.K., A.Y., and A.M.F. performed analyses (2–4). Thus far, transcriptome-wide profiling has been research; V.K., A.Y., T.L.M., and A.M.F. contributed new reagents/analytic tools; V.K., A.Y., reported for a limited number of modifications including T.L.M., A.M.F., C.J.B., and B.R.C. analyzed data; and V.K., C.J.B., and B.R.C. wrote N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudour- the paper. idine (Ψ), and N1-methyladenosine (m1A) (5–14). However, Reviewers: J.D.A., Ohio State University; T.C., Ludwig Maximilians University; and P.C.D., Massachusetts Institute of Technology. profiling methods that provide sensitive and true single-base The authors declare no conflict of interest. resolution are currently available only for m5C (9, 14, 15) and 1 6 1 Ψ This open access article is distributed under Creative Commons Attribution-NonCommercial- m A (16); three of these (m A, m A, and ) have involved initial NoDerivatives License 4.0 (CC BY-NC-ND). 6 1 enrichment or detection via antibodies (for m Aorm A) (5, 6, 8, Data deposition: The data reported in this paper have been deposited in the Gene Ex- 10) or by techniques involving polymerase pausing/termination pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. during reverse transcription (for m1A and Ψ) (7, 11–13, 17, 18). GSE90963). All custom computer scripts reported in this paper have been deposited in Recent single-base techniques for Ψ (19) rely on a bulky adduct GitHub, https://github.com/HuntsmanCancerInstitute/RBSSeqTools. formation before detection. Furthermore, although the current 1V.K. and A.Y. contributed equally to this work. methods for Ψ profiling are useful, most lack the sensitivity, 2Present address: Department of Stem Cells and Developmental Biology, Cell Science resolution, and technical ease needed for widespread adoption Research Center, Royan Institute for Stem Cell Biology and Technology, Academic – Center for Education, Culture and Research, 16635-148 Tehran, Iran. or straightforward candidate site validation (7, 11 13, 20). To 3 5 1 To whom correspondence may be addressed. Email: [email protected] or brad. provide simultaneous detection of m C, m A, and Ψ at single- [email protected]. base resolution transcriptome-wide from the same sample, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. developed a molecular approach and analysis pipelines for Ψ 1073/pnas.1817334116/-/DCSupplemental. 5 1 and improved sequencing-based methods for m C and m A. Published online March 14, 2019. 6784–6789 | PNAS | April 2, 2019 | vol. 116 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1817334116 together), 143 sites in mRNAs (e.g., PTEN and HDGF; Figs. 2 A and B and 3A, SI Appendix, Fig. S2A, and Datasets S1 and S2), 14 pseudogenes, and 32 other noncoding RNAs. New sites within prominent mRNAs include PTEN, XRCC3, FANCA, RXRB, FGFR4, and EIF3B (Dataset S1). Importantly, examination of known/validated sites in tRNAs demonstrated that RBS-Seq has a dynamic range for m5C approaching 100% at single cytosines (e.g., C49 in Fig. 1C). Although our read numbers exceeded prior studies, our yield of 486 high-threshold candidate m5C sites in mRNA was far lower than the 10,275 sites reported previously (14), largely due to our more effective denaturation and de- amination/conversion, lowering false positives (SI Appendix,Figs. S10 and S11,andDataset S1). In keeping, more recent m5Cpro- filing in mouse ESCs that applies additional statistical and ana- lytical parameters to remove false-positives reports 266 sites in mRNA passing thresholds (24). Unlike m6A, m1A compromises A:T Watson–Crick base pairing, which pauses reverse transcriptase and elicits frequent nucleotide misincorporation, generating a single-base mismatch signature useful for m1A identification (8, 17, 25). As expected, in our NBS datasets from RBS-Seq, we indeed detected significant (FDR < 0.01) m1A-related mismatches at well-known m1A sites in non- coding RNAs [e.g., m1A-1322 in 28S rRNA (Fig. 2C and SI Ap- pendix, Fig. S2B) and m1A-58 in all tRNAs (Fig. 2D and Dataset S3)]. Unexpectedly, these mismatches were wholly absent or greatly diminished in our BS datasets (SI Appendix,TableS3), with tRNAs displaying the remarkable dynamic range of our method (∼90% for tRNAGly and tRNAThr;Fig.2D). Regarding the basis, BIOCHEMISTRY 1 6 Fig. 1. conversion of m AtomA (involving transfer of the methyl group RBS-Seq enables simultaneous base-pair resolution transcriptome- 1 6 wide mapping of m5C, m1A, and Ψ.(A) Schematic of reactivity of modified from N to the N position) occurs through a well-studied process nucleotides to bisulfite (Top) and the principle of simultaneous mapping of known as the Dimroth rearrangement (26) (SI Appendix,Fig.S12), modified nucleotides (Bottom).
Load more

Recommended publications
  • How Shelterin Protects Mammalian Telomeres 303 ANRV361-GE42-15 ARI 3 October 2008 10:10 (See 3' 5' TRF2 S Mplex
    ANRV361-GE42-15 ARI 3 October 2008 10:10 ANNUAL How Shelterin Protects REVIEWS Further Click here for quick links to Annual Reviews content online, Mammalian Telomeres including: • Other articles in this volume 1 • Top cited articles Wilhelm Palm and Titia de Lange • Top downloaded articles • Our comprehensive search Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10021; email: [email protected] 1Current address: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany Annu. Rev. Genet. 2008. 42:301–34 Key Words First published online as a Review in Advance on ATM, ATR, cancer, NHEJ, HR August 4, 2008 by Rockefeller University on 10/13/09. For personal use only. The Annual Review of Genetics is online at Abstract genet.annualreviews.org The genomes of prokaryotes and eukaryotic organelles are usually cir- This article’s doi: cular as are most plasmids and viral genomes. In contrast, the nuclear Annu. Rev. Genet. 2008.42:301-334. Downloaded from arjournals.annualreviews.org 10.1146/annurev.genet.41.110306.130350 genomes of eukaryotes are organized on linear chromosomes, which Copyright c 2008 by Annual Reviews. require mechanisms to protect and replicate DNA ends. Eukaryotes All rights reserved navigate these problemswith the advent of telomeres, protective nucle- 0066-4197/08/1201-0301$20.00 oprotein complexes at the ends of linear chromosomes, and telomerase, the enzyme that maintains the DNA in these structures. Mammalian telomeres contain a specific protein complex, shelterin, that functions to protect chromosome ends from all aspects of the DNA damage re- sponse and regulates telomere maintenance by telomerase.
    [Show full text]
  • Synthetic Lethality Between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication
    Article Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication Graphical Abstract Authors Roberto Bellelli, Jillian Youds, Valerie Borel, Jennifer Svendsen, Visnja Pavicic-Kaltenbrunner, Simon J. Boulton Correspondence [email protected] In Brief Bellelli et al. report that RTEL1 deficiency is synthetic lethal with the loss of pole-4 in C. elegans/hypomorphy of Pol epsilon. An analysis of replication dynamics in Rtel1À/À Pole4À/À mouse cells revealed a combination of dysfunctional fork progression and defective origin activation, which cooperatively drive incomplete genomic replication and genetic instability. Highlights d rtel-1 is synthetic lethal with the loss of DNA polymerase epsilon in C. elegans d rtel-1; pole-4 double mutants accumulate Rad51 and RPA foci and fail to replicate d Impaired DNA replication and genome instability in Rtel1 Pole4 knockout mouse cells d Rtel1 Pole4 double knockout mouse cells exhibit fork asymmetry and defective origin activation Bellelli et al., 2020, Cell Reports 31, 107675 May 26, 2020 ª 2020 The Author(s). https://doi.org/10.1016/j.celrep.2020.107675 ll ll OPEN ACCESS Article Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication Roberto Bellelli,1,2,3 Jillian Youds,1,2 Valerie Borel,1 Jennifer Svendsen,1 Visnja Pavicic-Kaltenbrunner,1 and Simon J. Boulton1,4,* 1The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK 2These authors contributed equally 3Present address: Center for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, Barbican, EC1M 6BE London, UK 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2020.107675 SUMMARY Genome stability requires coordination of DNA replication origin activation and replication fork progression.
    [Show full text]
  • Dyskerin Mutations Present in Dyskeratosis Congenita Patients Increase Oxidative Stress and DNA Damage Signalling in Dictyostelium Discoideum
    cells Article Dyskerin Mutations Present in Dyskeratosis Congenita Patients Increase Oxidative Stress and DNA Damage Signalling in Dictyostelium Discoideum Javier Rodriguez-Centeno, Rosario Perona and Leandro Sastre * Instituto de Investigaciones Biomedicas, CSIC/UAM and Centro de Investigación Biomédica en Red de Enfermedades Raras, CIBERER, 28029 Madrid, Spain; [email protected] (J.R.-C.); [email protected] (R.P.) * Correspondence: [email protected]; Tel.: +3491-585-4437 Received: 11 October 2019; Accepted: 5 November 2019; Published: 8 November 2019 Abstract: Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease.
    [Show full text]
  • Single-Molecule Analysis of the Human Telomerase RNA Center Dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mu
    Single-Molecule Analysis of the Human Telomerase RNA center dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations Ashbridge, B; Orte, A; Yeoman, JA; Kirwan, M; Vulliamy, T; Dokal, I; Klenerman, D; Balasubramanian, S © 2009 American Chemical Society http://pubs.acs.org/page/policy/authorchoice_termsofuse.html For additional information about this publication click this link. http://qmro.qmul.ac.uk/xmlui/handle/123456789/14851 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] 10858 Biochemistry 2009, 48, 10858–10865 DOI: 10.1021/bi901373e Single-Molecule Analysis of the Human Telomerase RNA 3 Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations† Beth Ashbridge,‡,^ Angel Orte,‡,^,@ Justin A. Yeoman,‡,^,# Michael Kirwan, ) Tom Vulliamy, ) Inderjeet Dokal, ) David Klenerman,*,‡,r and Shankar Balasubramanian*,‡,§,r ‡University Chemical Laboratories, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., §School of Clinical Medicine, University of Cambridge, Cambridge CB2 0SP, U.K., and Centre) for Paediatrics, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AT, U.K. ^These authors contributed equally to this work. @Present address: Department of Physical Chemistry, Faculty of Pharmacy, Campus Cartuja, Granada 18071, Spain. #Present address: National Centre for Biological Sciences, Tata Institute of Fundamental Research, UAS GKVK, Bellary Road, Bangalore 560 065, India. rJoint senior authorship. Received August 7, 2009; Revised Manuscript Received October 1, 2009 ABSTRACT: It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme.
    [Show full text]
  • 1 POL30 Alleles in Saccharomyces Cerevisiae Reveal Complexities Of
    bioRxiv preprint doi: https://doi.org/10.1101/679829; this version posted June 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. POL30 alleles in Saccharomyces cerevisiae reveal complexities of the cell cycle and ploidy on heterochromatin assembly Molly Brothers and Jasper Rine Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 1 bioRxiv preprint doi: https://doi.org/10.1101/679829; this version posted June 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Running title: POL30 effect on heterochromatin assembly Keywords: PCNA, transcriptional silencing, recombination, nucleosome assembly, intragenic complementation Corresponding Author: Jasper Rine University of California, Berkeley, 16 Barker Hall #3220; (510) 642-7047; [email protected] 2 bioRxiv preprint doi: https://doi.org/10.1101/679829; this version posted June 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. ABSTRACT In Saccharomyces cerevisiae, transcriptional silencing at HML and HMR maintains mating-type identity. The repressive chromatin structure at these loci is replicated every cell cycle and must be re-established quickly to prevent transcription of the genes at these loci.
    [Show full text]
  • Chromosome Duplication in Saccharomyces Cerevisiae
    | YEASTBOOK GENOME ORGANIZATION AND INTEGRITY Chromosome Duplication in Saccharomyces cerevisiae Stephen P. Bell*,1 and Karim Labib†,1 *Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and yMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, DD1 5EH, United Kingdom ORCID ID: 0000-0002-2876-610X (S.P.B.) ABSTRACT The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. KEYWORDS DNA replication; cell cycle; chromatin; chromosome duplication; genome stability;
    [Show full text]
  • Family a and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions
    biology Review Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions Vinit Shanbhag 1,2, Shrikesh Sachdev 2,3, Jacqueline A. Flores 2,3, Mukund J. Modak 4 and Kamalendra Singh 2,3,4,5,* 1 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA; [email protected] 2 The Christopher S. Bond Life Science Center, University of Missouri, Columbia, MO 65211, USA; [email protected] (S.S.); [email protected] (J.A.F.) 3 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA 4 Department of Microbiology, Biochemistry and Molecular Genetics 225 Warren Street, NJ 07103, USA; [email protected] 5 Department of Laboratory Medicine, Karolinska Institutet, Stockholm 141 86, Sweden * Correspondence: [email protected]; Tel.: +1-573-882-9024 Received: 13 November 2017; Accepted: 29 December 2017; Published: 2 January 2018 Abstract: DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 30-50 exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes.
    [Show full text]
  • Microrna Modulate Alveolar Epithelial Response to Cyclic Stretch
    University of Pennsylvania ScholarlyCommons Departmental Papers (BE) Department of Bioengineering 2012 MicroRNA Modulate Alveolar Epithelial Response to Cyclic Stretch Nadir Yehya University of Pennsylvania, [email protected] Adi Yerrapureddy University of Pennsylvania John Tobias University of Pennsylvania, [email protected] Susan S. Margulies University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/be_papers Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Yehya, N., Yerrapureddy, A., Tobias, J., & Margulies, S. S. (2012). MicroRNA Modulate Alveolar Epithelial Response to Cyclic Stretch. BMC Genomics, 13 (154), http://dx.doi.org/10.1186/1471-2164-13-154 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/be_papers/205 For more information, please contact [email protected]. MicroRNA Modulate Alveolar Epithelial Response to Cyclic Stretch Abstract Background MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression implicated in multiple cellular processes. Cyclic stretch of alveoli is characteristic of mechanical ventilation, and is postulated to be partly responsible for the lung injury and inflammation in entilatv or-induced lung injury. We propose that miRNAs may regulate some of the stretch response, and therefore hypothesized that miRNAs would be differentially expressed between cyclically stretched and unstretched rat alveolar epithelial cells (RAECs). Results RAECs were isolated and cultured to express type I epithelial characteristics. They were then equibiaxially stretched to 25% change in surface area at 15 cycles/minute for 1 hour or 6 hours, or served as unstretched controls, and miRNAs were extracted. Expression profiling of the miRNAs with at least 1.5-fold change over controls revealed 42 miRNAs were regulated (34 up and 8 down) with stretch.
    [Show full text]
  • Qt38n028mr Nosplash A3e1d84
    ! ""! ACKNOWLEDGEMENTS I dedicate this thesis to my parents who inspired me to become a scientist through invigorating scientific discussions at the dinner table even when I was too young to understand what the hippocampus was. They also prepared me for the ups and downs of science and supported me through all of these experiences. I would like to thank my advisor Dr. Elizabeth Blackburn and my thesis committee members Dr. Eric Verdin, and Dr. Emmanuelle Passegue. Liz created a nurturing and supportive environment for me to explore my own ideas, while at the same time teaching me how to love science, test my questions, and of course provide endless ways to think about telomeres and telomerase. Eric and Emmanuelle both gave specific critical advice about the proper experiments for T cells and both volunteered their lab members for further critical advice. I always felt inspired with a sense of direction after thesis committee meetings. The Blackburn lab is full of smart and dedicated scientists whom I am thankful for their support. Specifically Dr. Shang Li and Dr. Brad Stohr for their stimulating scientific debates and “arguments.” Dr. Jue Lin, Dana Smith, Kyle Lapham, Dr. Tet Matsuguchi, and Kyle Jay for their friendships and discussions about what my data could possibly mean. Dr. Eva Samal for teaching me molecular biology techniques and putting up with my late night lab exercises. Beth Cimini for her expertise with microscopy, FACs, singing, and most of all for being a caring and supportive friend. Finally, I would like to thank Dr. Imke Listerman, my scientific partner for most of the breast cancer experiments.
    [Show full text]
  • Endosomal PI(3)P Regulation by the COMMD/CCDC22/CCDC93
    ARTICLE https://doi.org/10.1038/s41467-019-12221-6 OPEN Endosomal PI(3)P regulation by the COMMD/ CCDC22/CCDC93 (CCC) complex controls membrane protein recycling Amika Singla 1,5, Alina Fedoseienko2,5, Sai S.P. Giridharan3, Brittany L. Overlee2, Adam Lopez1, Da Jia 4, Jie Song1, Kayci Huff-Hardy1, Lois Weisman3, Ezra Burstein 1,6 & Daniel D. Billadeau2,6 1234567890():,; Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment. 1 Department of Internal Medicine, and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 2 Division of Oncology Research and Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
    [Show full text]
  • DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer
    cancers Review DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer Youri I. Pavlov 1,2,* , Anna S. Zhuk 3 and Elena I. Stepchenkova 2,4 1 Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Genetics and Biotechnology, Saint-Petersburg State University, 199034 Saint Petersburg, Russia; [email protected] 3 International Laboratory of Computer Technologies, ITMO University, 197101 Saint Petersburg, Russia; [email protected] 4 Laboratory of Mutagenesis and Genetic Toxicology, Vavilov Institute of General Genetics, Saint-Petersburg Branch, Russian Academy of Sciences, 199034 Saint Petersburg, Russia * Correspondence: [email protected] Received: 30 September 2020; Accepted: 13 November 2020; Published: 24 November 2020 Simple Summary: The etiology of cancer is linked to the occurrence of mutations during the reduplication of genetic material. Mutations leading to low replication fidelity are the culprits of many hereditary and sporadic cancers. The archetype of the current model of replication fork was proposed 30 years ago. In the sequel to our 2010 review with the words “years after” in the title inspired by A. Dumas’s novels, we go over new developments in the DNA replication field and analyze how they help elucidate the effects of the genetic variants of DNA polymerases on cancer. Abstract: Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization.
    [Show full text]
  • Download The
    DEVELOPMENT OF HUMAN-COMPUTER INTERACTIVE APPROACHES FOR RARE DISEASE GENOMICS by Jessica J. Y. Lee B.Sc., The University of British Columbia, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Genome Science and Technology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2018 © Jessica J. Y. Lee, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Development of Human-Computer Interactive Approaches for Rare Disease Genomics submitted by Jessica J. Y. Lee in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genome Science and Technology Examining Committee: Clara van Karnebeek, Pediatrics; Genome Science and Technology Co-supervisor Wyeth Wasserman, Medical Genetics Co-supervisor William Hsiao, Pathology and Laboratory Medicine Supervisory Committee Member Martin Dawes, Family Practice University Examiner Sabrina Wong, Nursing University Examiner Additional Supervisory Committee Members: Sara Mostafavi, Statistics Supervisory Committee Member Raymond Ng, Computer Science Supervisory Committee Member ii Abstract Clinical genome sequencing is becoming a tool for standard clinical practice. Many studies have presented sequencing as effective for both diagnosing and informing the management of genetic diseases. However, the task of finding the causal variant(s) of a rare genetic disease within an individual is often difficult due to the large number of identified variants and lack of direct evidence of causality. Current computational solutions harness existing genetic knowledge in order to infer the pathogenicity of the variant(s), as well as filter those unlikely to be pathogenic.
    [Show full text]