Fig. 1. DcpS activity elutes at the 40 kDa size range from an SDS–PAGE gel. MEL S130 extract (100 µg) was resolved by an SDS–PAGE and sequential gel slices were eluted and renatured as described (Rudolph and Lilie, 1996). The size and integrity of the protein in each sample were determined by rerunning an aliquot of the eluted protein on an SDS–PAGE gel and visualized by silver staining (data not shown). A sample of each eluted protein was tested for its ability to hydrolyze labeled cap analog and the products resolved by PEI TLC. The size range of the proteins is indicated at the top of each lane and migration of the standards visualized by ultraviolet shadowing is indicated on the left. Input, labeled cap analog is shown in lane 1. The schematic shown at the bottom represents the cap analog substrate used and the asterisk denotes the position of the labeled phosphate.

Fig. 2. Purification of DcpS. (A) The purification scheme is outlined including the columns and fractions used. Elution gradients are indicated by diagonal lines. The fractions containing DcpS activity are in bold. (B) Protein eluted from the final Blue HP column purification of DcpS protein from 150 l of MEL cells is shown. Proteins were eluted with an increasing KCl gradient from 100 to 670 mM and either resolved by SDS–PAGE and visualized by silver staining (top), or used in a decapping assay and the products resolved by PEI TLC (bottom). Labeling is as described in the legend to Figure 1. The decapping assays were carried out in the presence of 1 µM cold cap analog to slow the reaction. The fraction numbers are indicated between the two panels. The candidate band, which corresponded with DcpS activity, is indicated. Protein size markers are shown on the left in kDa.

Fig. 3. Recombinant DcpS is methyl-cap specific and does not hydrolyze capped RNA. (A) Total extract from E.coli BL21DE3 cells expressing His-tagged DcpS (lane 2) or purified recombinant protein (lane 3) resolved by SDS–PAGE and stained by Coomassie Blue are shown. The arrow denotes the His-tagged DcpS protein. Protein size markers are shown in lane 1 and the sizes indicated on the left. (B) His-tagged DcpS (100 ng) was tested for its ability to hydrolyze labeled cap analog (lanes 2–6). Lanes 3 and 4 contain 1 and 10 µM methylated cap analog competitor, while lanes 5 and 6 contain the same amount of unmethylated cap analog competitor. The substrate is shown at the bottom of the figure. (C) His-tagged DcpS was used to hydrolyze labeled cap analog (lane 2) or cap labeled RNA (lane 3). DcpS is unable to utilize the intact RNA as substrate. (D) Polyclonal antibody directed against recombinant His-DcpS was used to immunodeplete K562 S130 extract. A western blot of extract depleted with pre-immune or DcpS-specific antisera is shown in lanes 1 and 2, respectively. The products of a decapping assay using 10 µg of the respective depleted extract were resolved by PEI TLC (lanes 3 and 4). Migration of the standards and schematic of the substrate are indicated as above. Extract depleted of DcpS is devoid of scavenger decapping activity. (E) Decapping of labeled cap analog was carried out with His-tagged DcpS (100 ng) in the presence of the indicated competitors. Lanes 3, 6 and 9 contained 0.25 µM competitor. Lanes 4, 7 and 10 contained 1 µM competitor, and lanes 5, 8 and 11 contained 2.5 µM indicated competitor. The different levels of cap analog competitor susceptibility observed in (B) and (E) are due to the use of two separate preparations of recombinant DcpS for each panel.

Fig. 4. DcpS preferentially hydrolyzes RNAs <10 nucleotides. Recombinant DcpS (500 ng) was used in decapping assays utilizing various length cap-labeled substrates. Labeled cap analog (lane 1), which contains one nucleotide following the m⁷G, was used as the substrate in lane 2. 5′-end-labeled oligonucleotides of 10, 15 and 20 bases in length were used in lanes 3, 4 and 5, respectively. The decapping products were resolved by PEI TLC. Labeling is as described in the legend to Figure 1.

Fig. 5. The YLR270W gene encodes yeast DcpS (yDcpS). (A) Total yeast extract (50 µg) from the YLR270WΔ (lane 2), YOR173WΔ (lane 3) or wild-type cells (lane 4) was tested for the ability to hydrolyze labeled cap analog in a standard decay assay and the products resolved by PEI TLC. Extract from the YLR270WΔ strain lost the ability to hydrolyze cap analog (lane 2), while extract from the YOR173WΔ strain retains the ability to decap the same substrate. (B) The ability of His-tagged recombinant protein (500 ng) encoded by each yeast DcpS-like gene was used directly in an assay to determine their ability to cleave the labeled cap structure substrate. Consistent with the deletion strain data in (A) above, His-tagged protein expressed from the YLR270W gene was able to specifically hydrolyze the cap structure (lane 3), while protein expressed from YOR173W gene was not (lane 4). Purified His-tagged PARN was used as a negative control (lane 2). Labeling is as described in the legend to Figure 1. (C) Cap-labeled pcP RNA was electroporated into yeast cells and harvested 6 h post-electroporation and decapping products resolved by PEI TLC developed in 0.75 M LiCl to better resolve the lower spots. Decapping products from wild-type yeast cells (lane 1) and the yDcpSΔ strain (lane 2) are shown. The labeled cap analog standards were generated by treating cap-labeled pcP RNA with nuclease P1 (m⁷GpppG_OH; lane 3) or with RNase T1 (m⁷GpppG_p; lane 5). Migration of the ³²Pi-free phosphate is shown in lane 4. Products shown in lanes 1 and 2 were treated with recombinant yDcpS and resolved by TLC as shown in lanes 6 and 7, respectively. DcpS will hydrolyze cap analog present in these lanes to m⁷GMP.

Fig. 6. The DcpS HIT motif and its significance for decapping. (A) An alignment of the HIT hexapeptide (boxed region) and adjacent amino acids from potential DcpS homologs from different organisms is shown. The alignment was generated using the ClustalW alignment program with the default parameters. The sequences shown are: human DcpS (NP_054745), mouse DcpS (BAB24138), D.melanogaster DcpS (XP_082208), C.elegans DcpS (NP_507876), S.pombe DcpS (NP_596194), S.cerevisiae DcpS YLR270W (NP_013372) and the DcpS-like yeast protein YOR173W (NP_014816). The amino acid positions are indicated on the left and right of the sequences. Amino acids that are identical at a particular position in all seven proteins are shaded in black. Amino acids that are identical in four or more proteins at a specific position are shaded in gray. The position of the HIT hexapeptide is denoted by the bracket. (B) The significance of the DcpS HIT motif for decapping was tested by substitution of the central histidine at amino acid 277 with asparagine (H277N). Recombinant His-tagged wild-type (lane 3) or mutant protein (lane 4) was tested for the ability to hydrolyze labeled cap analog using 500 ng of protein. His-tagged PARN was used as a negative control (lane 2). Substitution of the central His ablates decapping (lane 4).