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Titolo:
Fending off decay: A combinatorial approach in intact cells for identifying mRNA stability elements
Autore:
Chrzanowska-Lightowlers, ZMA; Lightowlers, RN;
Indirizzi:
Univ Newcastle Upon Tyne, Sch Med, Dept Neurol, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England Univ Newcastle Upon Tyne Newcastle Upon Tyne Tyne & Wear England NE2 4HH
Titolo Testata:
RNA-A PUBLICATION OF THE RNA SOCIETY
fascicolo: 3, volume: 7, anno: 2001,
pagine: 435 - 444
SICI:
1355-8382(200103)7:3<435:FODACA>2.0.ZU;2-M
Fonte:
ISI
Lingua:
ENG
Soggetto:
MESSENGER-RNA STABILITY; POLY(C) BINDING-PROTEIN; 3' UNTRANSLATED REGION; CYTOCHROME-C OXIDASE; AU-RICH ELEMENTS; 3'-UNTRANSLATED REGION; TRANSLATIONAL EFFICIENCY; MAMMALIAN-CELLS; G-QUARTET; IDENTIFICATION;
Keywords:
GU-rich elements; mRNA stability; posttranscriptional regulation; selection;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
48
Recensione:
Indirizzi per estratti:
Indirizzo: Chrzanowska-Lightowlers, ZMA Univ Newcastle Upon Tyne, Sch Med, Dept Neurol, Farmlington Pl, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England Univ Newcastle Upon Tyne Farmlington Pl Newcastle Upon Tyne Tyne & Wear England NE2 4HH
Citazione:
Z.M.A. Chrzanowska-Lightowlers e R.N. Lightowlers, "Fending off decay: A combinatorial approach in intact cells for identifying mRNA stability elements", RNA, 7(3), 2001, pp. 435-444

Abstract

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however,a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells, Here, we report that this new methodology has been used in vivoto select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3 ' -untranslated region (UTR), downstreamfrom the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3 ' UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stabletranscripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3 ' UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/07/20 alle ore 21:22:16