The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping

Abstract

Eukaryotic mRNAs are modified at their 5′ end early during transcription by the addition of N 7-methylguanosine (m 7 G), 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 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 the yeast scavenger decapping enzyme 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.

Identification of high confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

“…First, we used the yeast scavenger decapping enzyme (yDcpS) 20,21 to remove the m 7 G cap from poly(A)-enriched RNA, leaving 5′-diphosphate ends. Second, the 5′-diphosphate RNA strands were recapped with 3′-azido-ddGTP using Vaccinia capping enzyme 21 (Supplementary Fig. 2).…”

“…Decapping and recapping of poly(A) RNA. Poly(A) RNA was decapped and recapped according to methods previously described 21 . In brief, decapping of 1.5-6 µg poly(A) RNA was performed with 1.5 µL yDcps (NEB, #M0463) in 1X yDcpS reaction buffer (10 mM Bis-Tris-HCl pH 6.5, 1 mM EDTA) in 50 µL total volume for 1 h at 37 ºC.…”

ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

“…Beyond the 7SL and 7SK examples, the systematic complementation of ReCappable-seq results with orthogonal datasets such as CAGE, can be used as a discovery platform to identify other interesting non-canonical capped structures. For example the trimethyl G cap is expected to be resistant to yDcpS [11] but captured by CAGE, leading to a discrepancy between ReCappable-seq and CAGE. Despite trimethyl G cap having been described on U1, U2, U4, and U5 snRNAs [38], ReCappable-seq identifies a clear pol-II consistent TSS at the start of these genes suggesting that only a fraction of the 5' end of these transcripts has been fully methylated to trimethyl G.…”

“…In order to include transcripts containing a canonical cap structure characteristic of Pol-II transcripts, we performed a prior decapping reaction to render capped RNA 5' ends amenable to capping by VCE. For this reaction, we used the property of the yDcpS decapping enzyme to hydrolyze the phosphodiester bond between the gamma and beta phosphates of the G-cap [11]. The reaction leaves a diphosphate-terminated 5' end that can be recapped by VCE with a biotinylated GTP derivative.…”

“…The reaction leaves a diphosphate-terminated 5' end that can be recapped by VCE with a biotinylated GTP derivative. Importantly, cap0 and cap1 as well as m7Gpppm6A and m7Gpppm6Am were all found to be suitable substrates for yDcpS [11] indicating that transcripts containing canonical G-cap structures can be recapped and sequenced.…”