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. 2003 Feb;9(2):231-8.
doi: 10.1261/rna.2151403.

Analysis of recombinant yeast decapping enzyme

Michelle Steiger  1 , Anne Carr-Schmid, David C Schwartz, Megerditch Kiledjian, Roy Parker
Affiliations

Analysis of recombinant yeast decapping enzyme

Michelle Steiger et al. RNA. 2003 Feb.

Abstract

A critical step in the turnover of yeast mRNAs is decapping. Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established. To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties. These experiments demonstrate that copurification of Dcp1p and Dcp2p yields active decapping enzyme under a variety of conditions. Moreover, Dcp2p alone can have decapping activity under some biochemical conditions. This suggests that Dcp2p can be a catalytic subunit of the decapping complex, and Dcp1p may function to enhance Dcp2p activity, or as an additional active subunit. In addition, recombinant Dcp1p/Dcp2p prefers long mRNA substrates and is sensitive to inhibition by sequestration of the 5' end but not the 3' end of the substrate. This suggests that Dcp1p/Dcp2p contains an additional RNA-binding site spatially distinct from the active site. Finally, using two RNA-binding proteins that enhance decapping in vivo (Edc1p and Edc2p), we can reconstitute the activation of decapping with recombinant proteins. This indicates that the Edc1 and Edc2 proteins act directly on the decapping enzyme.

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Figures

FIGURE 1.
FIGURE 1.
Purified fractions from cells expressing Dcp1p, Dcp2ΔCp, or Dcp1p and Dcp2ΔCp were assayed for decapping activity. Decapping reactions were carried out as described in Materials and Methods in 10 μL reactions with 1 fmole of MFA2 substrate and ∼20–30 ng of total purified protein. Reaction aliquots were taken and the reaction was stopped at the indicated time points; the reaction products were separated by PEI cellulose TLC. (−) Reaction with no enzyme present.
FIGURE 2.
FIGURE 2.
Decapping activity requires a normal Nudix motif in Dcp2ΔCp. Decapping assays analyzed the activity of purified protein fractions where Dcp2ΔCp is wild type or has the E153Q mutation. Dcp1p is present and wild type in all these protein preparations. Decapping reactions were carried out with 1 fmole MFA2 substrate in a 10-μL reaction and ∼225 ng of total protein in the purified fractions. Products were separated by PEI cellulose TLC. (−) Reaction with no enzyme present.
FIGURE 3.
FIGURE 3.
Decapping enzyme prefers longer substrates. A graphical representation of the activity observed with recombinant Dcp1p/Dcp2ΔCp decapping activity and different-sized MFA2 substrates. The sizes are indicated on the graph: 343 nucleotides (solid circles); 99 nucleotides (solid squares); 29 nucleotides (solid triangles); 20 nucleotides (open circles); and 1 nucleotide (cap analog, m7GpppG) (open triangles). Reactions were carried out as described in Materials and Methods with 1 fmole of the indicated MFA2 substrate, 10-μL reaction and 20 ng total protein. Products were separated by PEI cellulose TLC and quantitated with a Molecular Dynamics PhosphorImager.
FIGURE 4.
FIGURE 4.
Oligonucleotide Inhibition of decapping activity. The effects of adding oligonucleotides complementary to the substrate at different positions are shown following a decapping reaction (see Materials and Methods for sequences). The activity measurements indicated in the graph are normalized to the amount of activity in reactions with no oligo.
FIGURE 5.
FIGURE 5.
Edc proteins stimulate recombinant decapping activity. (A) Decapping reactions were carried out in a 15-μL reaction with Edc1p (8 fmoles), Edc2p (8 fmoles), or BSA present. In these reactions, a lower concentration of Dcp1p/Dcp2ΔCp was used to allow the stimulation of activity by Edc proteins to be visualized. The first panel is a PEI cellulose analysis of the reaction products generated after the indicated incubation time. (−) Reaction with no enzyme present. (B) A graphical representation of the data reactions with Edc2p (solid triangles), Edc1p (open circles), and no Edc protein but BSA (solid circles) with percent product plotted against time.
FIGURE 6.
FIGURE 6.
Mn2+ stimulates recombinant yeast decapping activity. (A) Decapping reactions were carried out by incubating 300 ng of the indicated histidine-tagged Dcp1p, Dcp2ΔCp, or Dcp2ΔCpQ153E proteins with cap-labeled pcP-G16RNA for 30 min at 37°C in our standard reaction buffer (Mg2+ only) or buffer supplemented with 2 mM MnCl2(Mg2+ and Mn2+). Standards were simultaneously developed and their positions are denoted on the right of the panel. (B) Gel-isolated Dcp2p is enzymatically active. Three micrograms of His-Dcp2 ΔC and Dcp2ΔC Q153E3 were resolved by a standard SDS/PAGE and visualized by Coomassie blue staining (left panel) or resolved in an identical gel containing cap-labeled pcP-G16 RNA and renatured and assayed for decapping activity (right panel) (see Materials and Methods). Protein size markers are indicated between the gels.
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