In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

Eukaryotic mRNAs are modified at their 5′ end early during transcription by the addition of N7-methylguanosine (m7G), which forms the cap on the first 5′ nucleotide. Identification of the 5′ nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effe...

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Published inbioRxiv
Main Authors Wulf, Madalee G, Buswell, John, Siu-Hong, Chan, Dai, Nan, Marks, Katherine, Martin, Evan R, Tzertzinis, George, Whipple, Joseph M, Correa, Ivan R, Schildkraut, Ira
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 20.05.2019
Subjects
Base composition
Enzymes
Guanosine
Nucleotides
Oligonucleotides
Sugar
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Summary:Eukaryotic mRNAs are modified at their 5′ end early during transcription by the addition of N7-methylguanosine (m7G), which forms the cap on the first 5′ nucleotide. Identification of the 5′ nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5′ end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.
DOI:10.1101/644492