Fending off decay: a combinatorial approach in intact cells for identifying mRNA stability elements.
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 vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3'-untranslated region (UTR), downstream from 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 stable transcripts. 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.
Chrzanowska-Lightowlers
Z
Z
Department of Neurology, University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne, United Kingdom. Z.Chrzanowska-Lightowlers@ncl.ac.uk
Lightowlers
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D020413:3' Untranslated Regions / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D018274:Electroporation; D017930:Genes, Reporter; D006207:Half-Life; D006801:Humans; D008099:Liver / Q000166:cytology; D008156:Luciferases / Q000235:genetics; D016254:Mutagenesis, Insertional; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D012641:Selection, Genetic
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D008156:Luciferases