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 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.

In a previous work, we developed a method termed Cappable-seq 9 to enrich primary prokaryotic RNA transcripts by capping their 5' triphosphate with 3'-desthiobiotin-GTP (DTB-GTP). In an effort to extend this method to eukaryotic mRNA, we demonstrate here that yDcpS can decap the 5' end of m 7 G-capped RNA transcripts of a length from 90 to 1400 nucleotides without appreciable length bias. Further, the yDcpS-treated transcripts can be recapped with DTB-GTP and recovered after binding to, and eluting from, streptavidin beads.

Results
Liu et al. 4 and Wypijewska et al. 10 presented data indicating that human DcpS (hDcpS) does not decap RNAs longer than approximately ten nucleotides. Cohen et al. showed that the nematode DcpS was even more limited in its ability to decap oligonucleotides longer than 3 nucleotides 11 . This precept of acting solely on capped short oligonucleotides has been attributed to all homologous HIT enzymes 12 , despite Salehi et al. 13 identifying a HIT protein in Schizosaccharomyces pombe, Nhm1, which was determined to decap long mRNA. Nhm1 has about 70% amino acid sequence similarity to the yeast and human DcpS enzymes. As these two views of Milac and Salehi are conflicting, we decided to characterize yDcpS with respect to its ability to decap RNA of different lengths. We used recombinant yDcpS expressed in E. coli and purified to homogeneity as described in Methods.

Pyrophosphorolysis of the m 7 GpppA dinucleotide
We first looked at catalysis of the dinucleotide cap analog m 7 GpppA, as the characterization of DcpS typically involves pyrophosphorolysis of cap analogs which are dinucleotides of NpppN structure 4,6,[14][15][16] . Cap analogs can be considered as a capped RNA of one nucleotide in length. Both Malys et al. 17 and Liu et al. 18 demonstrated the pyrophosphorolysis of m 7 GpppG dinucleotide with a rate constant of 0.012/sec for yDcpS and 0.09/sec for hDcpS, respectively. We compared the relative activity of hDcpS and yDcpS towards the m 7 GpppA dinucleotide. We determined the decapping rate at 50 µM m 7 GpppA dinucleotide by LC/MS analysis (Figure 2A). The results indicate the two enzymes catalyze the pyrophosphorolysis of dinucleotide with similar rates: yDcpS at 0.08/sec and hDcpS at 0.14/sec. Although the rate constant seen here for yDcpS is about 7 times faster than that reported by Malys et al., their incubation temperature was lower (30 ˚C), the ionic strength was greater (100 mM KOAc) and the pH was higher (pH 7.0). Thus, the catalysis of the dinucleotide observed with our preparation of yDcpS is consistent with published literature.

Yeast DcpS decaps a 25mer RNA
To determine whether yDcpS can decap an RNA longer than 15 nucleotides, a 25mer Cap 1 RNA synthesized and capped in vitro, was combined with total E. coli RNA and used as a substrate. The E. coli RNA was included to more closely resemble an in vitro reaction where only a subset of total eukaryotic RNA would be substrate for the DcpS. This mixture was incubated with yDcpS, and aliquots were sampled over time and analyzed by gel electrophoresis. As shown in Figure 2b, with increasing incubation time of up to three hours, a larger fraction of the 25mer RNA is shifted to the position of the decapped species, demonstrating that yDcpS is capable of decapping a longer RNA than what was reported previously.

The effect of ionic strength and pH on decapping activity
In vitro decapping of DcpS was assessed at various ionic strength and pH conditions by using a synthetic 25mer Cap 0 RNA modified with a 3'-FAM label. Substrate and product of the decapping reaction were resolved and quantified by capillary electrophoresis (CE) ( Figure 2c). As shown in Table 1A, the decapping reaction is significantly inhibited by increasing salt concentration, with KCl being a more potent inhibitor than NaCl. The optimal buffering pH was in the 6 -6.5 range. yDcpS exhibited very little decapping activity in the VCE Capping Buffer at pH 8.0 and in phosphate buffer at pH 7.0 (Table 1B).
Furthermore, a control reaction with a 5' triphosphate 3'-FAM labeled 25mer showed no measurable conversion of the triphosphate to di-or mono-phosphate after incubation with 50 µM yDcpS in decapping buffer at 37˚C for one hour as determined by CE analysis.

The effect of RNA 5' secondary structure on decapping accessibility
To determine whether the substrate secondary structure has an impact on the yDcpS activity, complementary synthetic RNA oligos were annealed to the 3'-FAM-labeled 25mer Cap 0 RNA to create a blunt end, a 10-nucleotide 5' recessed cap, or a 5-nucleotide 5' extended cap end. The extent of decapping was compared to the single-stranded capped 25mer. All 3 double-stranded substrates were decapped, with the 5' extended cap substrate being decapped as efficiently as the single-stranded control, while the 5' recessed and blunt ended caps were more resistant to decapping. These results indicate that while yDcpS prefers unstructured 5' ends, structured ends can also be decapped at higher enzyme concentration ( Figure 3) .

Cap specificity of yDcpS
Liu et al. 4 suggested that the cap analog GpppG was not likely a substrate for DcpS because the GpppG dinucleotide at 10 μM was not an effective inhibitor of m 7 GpppG decapping.
Wypijewska et al. 10  Various capped 25mer RNA substrates (100 nM) were incubated for 60 minutes at 37 °C with increasing concentrations of yDcpS varying by a factor of three. The reactions were then analyzed by PAGE and the relative extent of decapping determined after staining and imaging the gels. A typical set of gel images was compiled in Figure 4b. It can be estimated from the gel electrophoresis results that, m 7 GpppA, m 7 Gpppm 6 A, m 7 Gpppm 6 Am, m 7 GpppG, and m 7 GpppGm caps were removed at a similar yDcpS concentration of 0.9 µM, while GpppG required about a 3-fold higher concentration. Interestingly, while yDcpS can decap deoxyriboguanosine and arabinoguanosine caps, no activity was detected towards 2,2,7trimethylguanosine, adenosine, cytidine, or uridine capped RNAs ( Figure 4c). As for the length of the internucleotidic phosphate bridge, yDcpS was able to decap tri-or tetraphosphate, but no diphosphate caps. Not surprisingly, yDcpS had no activity on a nicotinamide adenine dinucleotide (NAD) capped RNA.

yDcpS decapping followed by VCE recapping
A characteristic of yDcpS catalysis is the removal of the m 7 G from the cap in the form of m 7 GMP leaving a 5'-diphosphate on the 5' terminus of the RNA 4 . 5'-diphosphate RNA is a known substrate for in vitro capping using the Vaccinia virus Capping Enzyme (VCE) 9,24,25 .
We took advantage of these two reactions to tag a m 7 G-capped 25mer RNA with an affinity group, by first decapping the RNA with yDcpS, and then recapping it by treatment with VCE and DTB-GTP 9 . These reactions were performed in the presence of excess total E. coli RNA in order to simulate a complex mixture of RNA as would be found in total eukaryotic RNA.
As shown by gel electrophoresis for a Cap 1 RNA ( Figure 5) and by mass spectroscopy for Cap 0 (Supplementary Fig. S1-S3), the decapping/recapping process approached completion, suggesting that this strategy could be extended to capture and enrich native capped RNA transcripts.

yDcpS decaps long capped RNA transcripts
After demonstrating the efficient decapping and recapping of m 7 G-capped 25mer RNAs, we tested whether yDcpS could decap long RNA substrates. We generated a mixture of RNA transcripts with lengths of 90 to 1400 nucleotides by in vitro transcription from a plasmid harboring a T7 promoter upstream of the FLuc gene, which had been cleaved with various restriction endonucleases to generate transcription templates of different lengths (see Methods). The produced RNA transcripts were capped with GTP by VCE to yield m 7 Gcapped RNAs. The mixture of capped transcripts was then decapped by incubation with yDcpS. As a control, an aliquot of the m 7 G-capped transcripts was incubated in the absence of yDcpS. Both sets of transcripts were subjected to capping with DTB-GTP by VCE. The recapped products were exposed to streptavidin beads, and after washing steps to remove any unbound material, recovered by elution with biotin. As shown in Figure 6, no RNA was recovered from the fraction that was not treated with yDcpS (Lane 4), while the full-size range of capped transcripts treated with yDcpS were recovered in the biotin-eluted fraction. This result demonstrates that yDcpS decapping followed by VCE recapping and streptavidin-based recovery of transcripts is an efficient process (approximately 50% yield) and shows minimal length discrimination. This workflow can thus be applied to eukaryotic mRNA Transcription Start Site (TSS) analysis by appending it to the workflow of Cappable-seq 9 .

Discussion
In characterizing yDcpS, we have shown that the methylation status of various canonical guanosine caps (GpppN, m 7 GpppN, m 7 GpppNm, m 7 Gpppm 6 A, or m 7 Gpppm 6 Am) does not significantly affect their susceptibility to decapping by this enzyme. We also examined noncanonical m 7 G caps where the ribose moiety was substituted with arabinose or deoxyribose. Although these caps have not been shown to exist in nature, they are also decapped by yDcpS.
Under the conditions described, yDcpS decaps the majority of the guanosine-based caps, regardless of the nucleotide at position +1. The enzyme prefers m 7 G over nonmethylated G caps, and does not decap adenosine, cytosine, uridine, or inosine caps. yDcpS decaps 5'-5' triphosphate and tetraphosphate linkages, but not diphosphate linkages. Interestingly, Cohen et al. 11 reported that nematode DcpS decapped a 2,2,7-trimethylated G cap (commonly found in certain small nuclear and nucleolar RNAs), while the human DcpS did not. We found that yDcpS, resembling hDcpS, did not decap 2,2,7-trimethylated guanosine.
The in vitro characterization of yDcpS presented here provides evidence that yDcpS should be a useful reagent for generating recappable 5' ends from capped RNAs. We demonstrate that yDcpS decaps m 7 G-capped RNAs from as short as 25 to at least 1400 nucleotides, leaving a 5'-diphosphate end. The process of decapping and recapping with an affinitytagged nucleotide would be advantageous for identifying and enriching 5' capped termini of mRNAs in eukaryotes. This would expand the utility of the prokaryotic Cappable-seq method 9 to include eukaryotic mRNAs. Cappable-seq has been used to determine the transcription start sites of primary transcripts of prokaryotes at single base resolution via the attachment of a biotinylated GTP analogue to the 5'-triphosphate end of primary RNA transcripts by action of the Vaccinia capping enzyme (VCE). The extension of this method to canonical eukaryotic mRNAs requires the removal of the m 7 G moiety to generate a recappable end (such as a tri-or diphosphate 5' end), which we now show is attainable with the use of yDcpS. Therefore, we anticipate being able to enrich eukaryotic mRNA by combining yDcpS-mediated decapping followed by recapping with affinity tagged GTP and generating libraries for high-throughput sequencing. Progress towards this goal is underway in our laboratory.

Preparation of yDcpS
The gene for the Saccharomyces cerevisiae yDcpS (YLR270W) was codon optimized for E. coli expression and designed with an amino terminal His tag to be expressed from a T7 promoter in plasmid pET28a (Supplementary Fig. S4). The predicted molecular weight of the fusion is 42,945 daltons. The construct was synthesized by Genscript. The protein was expressed in E. coli ER3600 and purified to near homogeneity (greater than 95%) by chromatography over HisTrap, SP, and Q resins. The protein's size as determined by SDS-PAGE is consistent with its predicted molecular weight. The final preparation of yDcpS was 12 µg of protein/µL, in a storage buffer of 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.1 mM EDTA, and 50% glycerol. The protein was stored at -20 ˚C. The DcpS preparation was determined to be free of detectable RNAse contamination by assay with a 300mer RNA transcript (~100 ng) incubated with 12 µg of yDcpS in a 20 µL reaction in the decapping buffer 10 mM Bis-Tris pH 6.5, 1 mM EDTA , and in 20 mM Tris-Acetate pH 7.9 buffer containing 50 mM potassium acetate, 10 mM magnesium acetate, and 1 mM DTT for 4 hours. No degradation of the 300mer band by PAGE analysis was observed.

Human DcpS
Human DcpS (hDcpS) was purchased from Enzymax LLC, Lexington, KY. The protein concentration of DcpS was determined by OD280 using the molar extinction coefficient of 30495 M -1 cm -1 .

Decapping reactions
Unless otherwise noted, the decapping was carried out at 37 ˚C in the decapping buffer 10 mM Bis-Tris pH 6.5, 1 mM EDTA. With the exception of the reactions containing FAMlabeled RNAs (see below), all reactions using capped 25mer RNA substrates were terminated by the addition of 1 volume of 2X RNA loading dye (NEB). The reactions using the m 7 GpppA dinucleotide cap analog were terminated with the addition of phenol/chloroform.

In vitro decapping of 5'-capped synthetic 3'-FAM-labeled RNA
In vitro decapping reactions were carried out in a 20 µL reaction containing 10 mM Bis-Tris To each 12 µL reaction was added 12 µL of RNA loading dye (2X) (NEB). This mixture was heat denatured at 80 ˚C for 3 minutes, and an aliquot of 5 µL was loaded onto a 15% polyacrylamide TBE-Urea gel (Invitrogen). The gel was run at 180 volts for 75 minutes. The RNA was visualized with SYBR Gold stain.

Decapping 25mer with yDcpS and recapping with 3'-Desthiobiotin-GTP
A 100 µL reaction in 10 mM MES pH 6.5 and 1 mM EDTA containing 0.22 μg of 25mer Cap1 RNA and 0.1 nmol of yDcpS, and 1 µg of total E. coli RNA, was incubated for 2 hours at 37 ˚C.
Before addition of the yDcpS, a 2 µL sample of the reaction was removed. The yDcpS reaction was terminated by addition of 4 µL of 1M Tris HCl pH 8.0 and 2 µL of Proteinase K.
The reaction was further incubated for 30 minutes at 37 ˚C and then heated to 94 ˚C for 3 minutes. The RNA was purified by binding to Ampure XP beads (Beckman Coulter) by addition of 2 volumes of beads, and to that final volume additional 1.5 volumes of ethanol.
After washing the beads with 80% ethanol, the RNA was eluted in 30 µL of 1 mM Tris pH

Data acquisition and settings
RNA was loaded onto 6% or 15% PAGE Urea gels and electrophoresed for 75-85 minutes at 180 V. The gels were stained with SYBR Gold and imaged with an AlphaImager HP. The images were imported into Photoshop, inverted and levels were auto-adjusted.      Substrates decapped (greater than 95% at 2.7 µM yDcpS) are shaded in green and substrates resistant to decapping (less than 10% at 25 µM yDcpS) are shaded in pink.
Bolded characters indicate canonical cap structures (known or anticipated). Asterisks indicate 5-10 fold reduced decapping relative to m7GpppG-25mer at 0.9 μM yDcpS.  Efficient recovery of long capped RNA transcripts by a decapping/recapping procedure. A mixture of m 7 G-capped RNAs (0.090 -1.4 kb) was divided into two samples. One sample was incubated with yDcpS and one without yDcpS, and both were subsequently treated with VCE and DTB-GTP. An equal fraction of each was exposed to streptavidin beads, which were washed and eluted. Lanes 1 (+ yDcpS) and 2 (-yDcpS) show the samples after the VCE reaction. Lanes 3 (+ yDcpS) and 4 (-yDcpS) show the eluates from streptavidin beads. All lanes represent an equal fraction of the original mixture. Both gel panels are from the same imaged gel.