Design and Delivery of SINEUP: A New Modular Tool to Increase Protein Translation.
Antisense
Haploinsufficiency
Long non-coding RNA
Physiological increase
Protein manufacturing
SINEUP
Therapeutic tool
Translational increase
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
25
2
2022
pubmed:
26
2
2022
medline:
3
3
2022
Statut:
ppublish
Résumé
SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouse AS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)-containing the invSINEB2 element-able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.
Identifiants
pubmed: 35213010
doi: 10.1007/978-1-0716-2010-6_4
pmc: PMC9703201
doi:
Substances chimiques
RNA, Antisense
0
RNA, Long Noncoding
0
RNA, Messenger
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
63-87Informations de copyright
© 2022. The Author(s).
Références
Front Cell Neurosci. 2015 May 13;9:174
pubmed: 26029048
Algorithms Mol Biol. 2011 Nov 24;6:26
pubmed: 22115189
Comput Struct Biotechnol J. 2016 Oct 27;14:404-410
pubmed: 27872686
Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W70-4
pubmed: 18424795
Discov Med. 2014 Jul-Aug;18(97):67-77
pubmed: 25091489
PLoS One. 2018 Feb 7;13(2):e0183229
pubmed: 29414979
PLoS One. 2011;6(10):e26203
pubmed: 22022567
Nat Biotechnol. 2014 Mar;32(3):217-9
pubmed: 24727769
MAbs. 2018 Jul;10(5):730-737
pubmed: 29658818
RNA Biol. 2015;12(8):771-9
pubmed: 26259533
FEBS Lett. 2020 Dec;594(24):4357-4369
pubmed: 33012004
Nat Protoc. 2016 Feb;11(2):273-90
pubmed: 26766114
Sci Rep. 2016 Jun 06;6:27315
pubmed: 27265476
Gene. 2002 Oct 16;299(1-2):1-34
pubmed: 12459250
Nat Rev Drug Discov. 2013 Jun;12(6):433-46
pubmed: 23722346
Nucleic Acids Res. 2020 Sep 18;48(16):9346-9360
pubmed: 32697302
J Vis Exp. 2019 Feb 1;(144):
pubmed: 30774120
Sci Rep. 2016 Sep 20;6:33605
pubmed: 27646849
Nature. 2012 Nov 15;491(7424):454-7
pubmed: 23064229
Nucleic Acids Res. 2019 Nov 18;47(20):10728-10743
pubmed: 31584077
Adv Exp Med Biol. 2017;983:1-20
pubmed: 28639188
Methods Mol Biol. 2016;1434:29-43
pubmed: 27300529
Mol Ther. 2020 Feb 5;28(2):642-652
pubmed: 31495777
Gene. 2015 Sep 15;569(2):287-93
pubmed: 26045368
Nucleic Acids Res. 2015 May 19;43(9):e58
pubmed: 25722369