Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
18 03 2021
18 03 2021
Historique:
accepted:
20
11
2020
revised:
16
11
2020
received:
30
07
2020
pubmed:
18
12
2020
medline:
27
3
2021
entrez:
17
12
2020
Statut:
ppublish
Résumé
Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation.
Identifiants
pubmed: 33330921
pii: 6039916
doi: 10.1093/nar/gkaa1185
pmc: PMC7969011
doi:
Substances chimiques
RNA, Messenger
0
Arginine
94ZLA3W45F
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e26Subventions
Organisme : NCI NIH HHS
ID : P30 CA008748
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM121847
Pays : United States
Informations de copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
Références
BMC Biol. 2019 Dec 6;17(1):101
pubmed: 31810458
Annu Rev Biophys. 2015;44:143-66
pubmed: 25747594
EMBO J. 2001 Nov 15;20(22):6453-63
pubmed: 11707416
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
pubmed: 12824337
Structure. 2013 Dec 3;21(12):2197-207
pubmed: 24183571
Nucleic Acids Res. 2008 May;36(8):2476-88
pubmed: 18310103
EMBO J. 2020 Feb 3;39(3):e103365
pubmed: 31858614
Nature. 2018 Jul;559(7712):130-134
pubmed: 29950728
Nucleic Acids Res. 2012 Jul;40(13):6199-207
pubmed: 22457067
Cold Spring Harb Perspect Biol. 2019 Feb 1;11(2):
pubmed: 29891560
Mol Cell. 2015 Nov 5;60(3):475-86
pubmed: 26593721
Methods Enzymol. 2014;539:17-28
pubmed: 24581436
Science. 1962 Dec 28;138(3548):1399-403
pubmed: 17753859
J Biol Chem. 2010 Apr 23;285(17):12755-64
pubmed: 20159983
Mol Biol Cell. 1999 Nov;10(11):3971-8
pubmed: 10564284
RNA. 2006 Jul;12(7):1338-49
pubmed: 16682564
J Mol Biol. 2006 Jul 21;360(4):893-906
pubmed: 16784753
Mol Cell. 2010 Oct 8;40(1):138-46
pubmed: 20932481
Chem Soc Rev. 2014 Feb 21;43(4):1044-56
pubmed: 24177677
Nature. 1994 Aug 18;370(6490):578-81
pubmed: 8052314
Mol Cell Biol. 1998 Dec;18(12):7528-36
pubmed: 9819438
Cell. 2011 Jun 10;145(6):902-13
pubmed: 21663794
Biochem Soc Trans. 2018 Jun 19;46(3):503-512
pubmed: 29626148
Mol Cell Biol. 1997 Sep;17(9):4904-13
pubmed: 9271370
Annu Rev Microbiol. 2005;59:407-50
pubmed: 16153175
PLoS Genet. 2013;9(8):e1003529
pubmed: 23950723
EMBO J. 2016 Apr 1;35(7):706-23
pubmed: 26896445
Mol Cell. 2016 Apr 7;62(1):92-103
pubmed: 27058789
Proc Natl Acad Sci U S A. 1963 May;49(5):654-62
pubmed: 16591084
ACS Med Chem Lett. 2018 Nov 21;9(12):1285-1291
pubmed: 30613341
Cell. 1987 Jun 19;49(6):805-13
pubmed: 3555844
Nucleic Acids Res. 2015 Jan;43(1):618-28
pubmed: 25520190
J Neurosci Methods. 1991 Nov;40(1):71-86
pubmed: 1795554
J Biol Chem. 1997 Jan 3;272(1):255-61
pubmed: 8995256
Nat Struct Mol Biol. 2019 Jun;26(6):501-509
pubmed: 31160784
Proc Natl Acad Sci U S A. 2017 Dec 19;114(51):E10899-E10908
pubmed: 29208708
Science. 2009 Apr 10;324(5924):218-23
pubmed: 19213877
J Biol Chem. 2016 Mar 18;291(12):6546-58
pubmed: 26817837
Annu Rev Biochem. 2013;82:171-202
pubmed: 23746254
Trends Biochem Sci. 2019 Sep;44(9):782-794
pubmed: 31003826
RNA. 2010 Sep;16(9):1832-47
pubmed: 20675403
Int J Biochem Cell Biol. 2013 Aug;45(8):1690-700
pubmed: 23624144
J Biol Chem. 2001 Oct 12;276(41):38036-43
pubmed: 11489903
Cell. 2006 Jun 16;125(6):1095-109
pubmed: 16777600
Mol Cell Biol. 2012 Jul;32(13):2396-406
pubmed: 22508989
EMBO J. 1988 Nov;7(11):3559-69
pubmed: 2850168
Mol Cell. 2005 Nov 11;20(3):449-60
pubmed: 16285926
Mol Cell Biol. 1996 Feb;16(2):603-8
pubmed: 8552088
Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):E2424-32
pubmed: 22927429
J Biol Chem. 1999 Dec 31;274(53):37565-74
pubmed: 10608810
Science. 2000 Aug 11;289(5481):920-30
pubmed: 10937990
Nat Rev Genet. 2011 Aug 31;12(10):683-91
pubmed: 21878961
Nature. 2004 Nov 4;432(7013):112-8
pubmed: 15525991
Yeast. 1999 Jun 15;15(8):633-7
pubmed: 10392441
Nature. 1963 Feb 2;197:430-5
pubmed: 14000153
Mol Cell. 2018 Apr 19;70(2):254-264.e6
pubmed: 29677493
Nat Struct Mol Biol. 2019 Dec;26(12):1132-1140
pubmed: 31768042
J Phys Chem B. 2010 Jan 14;114(1):280-92
pubmed: 20000829
Mol Cell Biol. 1998 Jan;18(1):51-7
pubmed: 9418852
Cold Spring Harb Perspect Biol. 2018 Nov 1;10(11):
pubmed: 30385605
Cell Rep. 2014 Nov 20;9(4):1256-64
pubmed: 25456128
Nat Rev Genet. 2011 Jan;12(1):32-42
pubmed: 21102527
Fungal Genet Biol. 2007 Feb;44(2):93-104
pubmed: 16979358
Nature. 2011 Jul 06;475(7354):118-21
pubmed: 21734708
Trends Biochem Sci. 2017 Aug;42(8):589-611
pubmed: 28442192
Biophys J. 2015 Dec 1;109(11):2352-62
pubmed: 26636946
Proc Natl Acad Sci U S A. 1963 Jan 15;49:122-9
pubmed: 13998950
J Vis Exp. 2020 Sep 15;(163):
pubmed: 33016943
Nucleic Acids Res. 2017 Sep 6;45(15):8844-8858
pubmed: 28637336
Nucleic Acids Res. 2020 Jan 10;48(1):e6
pubmed: 31722415
Genetics. 2016 May;203(1):65-107
pubmed: 27183566