دورية أكاديمية
أعرض في EDS

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.

التفاصيل البيبلوغرافية
العنوان: Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.
المؤلفون: Smolka JA; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA., Sanz LA; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA., Hartono SR; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA., Chédin F; Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA.
المصدر: The Journal of cell biology [J Cell Biol] 2021 Jun 07; Vol. 220 (6).
نوع المنشور: Journal Article; Research Support, N.I.H., Extramural
اللغة: English
بيانات الدورية: Publisher: Rockefeller University Press Country of Publication: United States NLM ID: 0375356 Publication Model: Print Cited Medium: Internet ISSN: 1540-8140 (Electronic) Linking ISSN: 00219525 NLM ISO Abbreviation: J Cell Biol Subsets: MEDLINE
أسماء مطبوعة: Original Publication: New York : Rockefeller University Press
مواضيع طبية MeSH: Antibodies, Monoclonal/*metabolism , DNA/*metabolism , Nucleic Acid Heteroduplexes/*metabolism , RNA/*metabolism , Ribonuclease H/*metabolism, Antibodies, Monoclonal/chemistry ; Antibody Affinity ; Artifacts ; DNA/chemistry ; Humans ; Nucleic Acid Heteroduplexes/chemistry ; RNA/chemistry ; Ribonuclease H/chemistry
مستخلص: The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.
(© 2021 Smolka et al.)
References: Dis Model Mech. 2018 Feb 26;11(2):. (PMID: 29419415)
Nature. 2014 Dec 18;516(7531):436-9. (PMID: 25296254)
DNA Repair (Amst). 2019 Dec;84:102672. (PMID: 31371183)
Mol Cell. 2009 Jul 31;35(2):228-39. (PMID: 19647519)
Mol Cell. 2018 Dec 20;72(6):970-984.e7. (PMID: 30449723)
Noncoding RNA. 2018 Mar 27;4(2):. (PMID: 29657305)
Oncogene. 2018 May;37(18):2351-2366. (PMID: 29429989)
PLoS Genet. 2014 Oct 30;10(10):e1004716. (PMID: 25357144)
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3073-8. (PMID: 10737787)
Nat Genet. 2016 Nov;48(11):1385-1395. (PMID: 27668659)
Nat Commun. 2017 Aug 18;8(1):283. (PMID: 28819201)
Mol Cell. 2012 Mar 30;45(6):814-25. (PMID: 22387027)
Curr Opin Cell Biol. 2018 Jun;52:105-111. (PMID: 29529563)
Nucleic Acids Res. 2017 Oct 13;45(18):10672-10692. (PMID: 28977560)
Nat Plants. 2017 Sep;3(9):704-714. (PMID: 28848233)
EMBO Rep. 2019 Sep;20(9):e47250. (PMID: 31338941)
J Mol Recognit. 2013 Aug;26(8):376-81. (PMID: 23784994)
Cell Rep. 2018 May 8;23(6):1891-1905. (PMID: 29742442)
J Biol Chem. 2014 Nov 14;289(46):31827-31836. (PMID: 25246524)
Nature. 2014 Jul 17;511(7509):362-5. (PMID: 24896180)
Genes Dev. 2012 Jan 15;26(2):163-75. (PMID: 22279048)
Mol Cell. 2017 Mar 2;65(5):832-847.e4. (PMID: 28257700)
Genes Dev. 2014 Jul 1;28(13):1384-96. (PMID: 24990962)
Proc Natl Acad Sci U S A. 2019 Jan 15;116(3):816-825. (PMID: 30591567)
Mol Cell. 2011 Jun 24;42(6):794-805. (PMID: 21700224)
EMBO J. 2019 Aug 1;38(15):e100986. (PMID: 31267554)
Nat Genet. 2020 Jan;52(1):48-55. (PMID: 31844323)
Cell Rep. 2017 Jan 10;18(2):334-343. (PMID: 28076779)
Mol Cell. 2016 Jul 7;63(1):167-78. (PMID: 27373332)
Nat Chem Biol. 2020 Jan;16(1):50-59. (PMID: 31819276)
Mol Cell. 2003 Sep;12(3):711-21. (PMID: 14527416)
Trends Genet. 2016 Dec;32(12):828-838. (PMID: 27793359)
Mol Cell. 2014 Dec 18;56(6):777-85. (PMID: 25435140)
Nat Protoc. 2019 Jun;14(6):1734-1755. (PMID: 31053798)
Proc Natl Acad Sci U S A. 2018 Oct 23;115(43):11024-11029. (PMID: 30301808)
PLoS Genet. 2014 Apr 17;10(4):e1004294. (PMID: 24743386)
Mol Cell. 2012 Apr 27;46(2):115-24. (PMID: 22541554)
Mol Cell. 2015 Feb 19;57(4):636-647. (PMID: 25699710)
J Biol Chem. 2015 Mar 20;290(12):7463-73. (PMID: 25623070)
Nucleic Acids Res. 2019 Jun 20;47(11):5480-5489. (PMID: 31045202)
Science. 2018 Jan 5;359(6371):108-114. (PMID: 29170278)
Genes Dev. 2010 Jul 15;24(14):1546-58. (PMID: 20634320)
Nat Methods. 2012 Mar 04;9(4):357-9. (PMID: 22388286)
Genome Biol. 2008;9(9):R137. (PMID: 18798982)
Nat Chem Biol. 2007 Sep;3(9):570-5. (PMID: 17643112)
EMBO J. 2014 Mar 18;33(6):542-58. (PMID: 24514026)
Mol Cell. 2019 Feb 7;73(3):398-411. (PMID: 30735654)
Nucleic Acids Res. 2013 Dec;41(22):10110-23. (PMID: 23999093)
J Clin Microbiol. 1989 Jan;27(1):6-12. (PMID: 2536393)
Cancer Res. 2018 Sep 15;78(18):5363-5374. (PMID: 30054334)
PLoS One. 2008;3(11):e3716. (PMID: 19005571)
Nucleic Acids Res. 2018 Feb 28;46(4):1912-1926. (PMID: 29315404)
Cell. 2005 Aug 12;122(3):365-78. (PMID: 16096057)
EMBO J. 2021 Feb 15;40(4):e106394. (PMID: 33411340)
J Immunol Methods. 1986 May 1;89(1):123-30. (PMID: 2422282)
Cell Stress. 2018 May 10;2(6):125-140. (PMID: 31225478)
J Mol Biol. 2018 Feb 2;430(3):272-284. (PMID: 29289567)
Elife. 2013 Jun 11;2:e00505. (PMID: 23795288)
Genome Res. 2018 Sep;28(9):1405-1414. (PMID: 30108179)
Cell. 2017 Aug 10;170(4):774-786.e19. (PMID: 28802045)
Nucleic Acids Res. 2020 Aug 20;48(14):e84. (PMID: 32544226)
EMBO J. 2017 May 2;36(9):1182-1198. (PMID: 28314779)
معلومات مُعتمدة: R01 GM120607 United States GM NIGMS NIH HHS; T32 GM007377 United States GM NIGMS NIH HHS
المشرفين على المادة: 0 (Antibodies, Monoclonal)
0 (Nucleic Acid Heteroduplexes)
63231-63-0 (RNA)
9007-49-2 (DNA)
EC 3.1.26.4 (Ribonuclease H)
EC 3.1.26.4 (ribonuclease HI)
تواريخ الأحداث: Date Created: 20210408 Date Completed: 20211011 Latest Revision: 20211208
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC8040515
DOI: 10.1083/jcb.202004079
PMID: 33830170
قاعدة البيانات: MEDLINE
الوصف
تدمد:1540-8140
DOI:10.1083/jcb.202004079