Analysis of recombinant yeast decapping enzyme.
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.
Steiger
Michelle
M
Department of Molecular and Cellular Biology & Howard Hughes Medical Institute, University of Arizona, 1007 E. Lowell Street, Tucson, AZ 85704, USA.
Carr-Schmid
Anne
A
Schwartz
David C
DC
Kiledjian
Megerditch
M
Parker
Roy
R
D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology* / Q000378:metabolism
C421609:Edc1 protein, S cerevisiae; C421610:Edc2 protein, S cerevisiae; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C030052:mRNA decapping enzymes; D012313:RNA; C100868:DCP1 protein, S cerevisiae; C400572:DCP2 protein, S cerevisiae; D004722:Endoribonucleases