The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1

Abstract

An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m(7)G cap structure at the 5' end of the message. Here, we describe the functional characterization of Dhh1, a highly conserved member of the family of DEAD box-containing proteins, as a regulator of mRNA decapping in Saccharomyces cerevisiae. Dhh1 is a cytoplasmic protein and is shown to be in a complex with the mRNA degradation factor Pat1/Mtr1 and with the 5'-3' exoribonuclease Xrn1. Dhh1 specifically affects mRNA turnover in the deadenylation-dependent decay pathway, but does not act on the degradation of nonsense-containing mRNAs. Cells that lack dhh1 accumulate degradation intermediates that have lost their poly(A) tail but contain an intact 5' cap structure, suggesting that Dhh1 is required for efficient decapping in vivo. Furthermore, recombinant Dhh1 is able to stimulate the activity of the purified decapping enzyme Dcp1 in an in vitro decapping assay. We propose that the DEAD box protein Dhh1 regulates the access of the decapping enzyme to the m(7)G cap by modulating the structure at the 5' end of mRNAs.

Fig. 1. (A) Dhh1 is a cytoplasmic protein. The subcellular localization of the Dhh1 protein was analyzed by indirect immunofluorescence using a polyclonal anti-Dhh1 serum. Cells were stained either with anti-Dhh1 antiserum and anti-rabbit (FITC) antibody (top panel) or only with anti-rabbit (FITC) antibody (lower panel). The right panel shows the corresponding DNA (DAPI) staining. (B) Purification of Dhh1-interacting proteins. Dhh1-interacting proteins were purified from Dhh1–ZZ-expressing cells by IgG affinity chromatography. Proteins were eluted using 1 M MgCl₂, separated on an 8% SDS gel and stained with Coomassie Blue. Proteins indicated by an arrow were identified by MALDI-TOF mass spectrometry. Thirty-six peptides out of 69 were matching peptides for Xrn1 (21% sequence coverage), 24 peptides out of 43 matched Pat1 (24% sequence coverage). Molecular weight markers are indicated on the left. The names of the proteins identified by MALDI are listed on the right. Xrn1* indicates a degradation product of Xrn1. Bands that are not specific for Dhh1p interaction are not labeled. As a control strain the isogenic wild-type background was used.

Fig. 1. (A) Dhh1 is a cytoplasmic protein. The subcellular localization of the Dhh1 protein was analyzed by indirect immunofluorescence using a polyclonal anti-Dhh1 serum. Cells were stained either with anti-Dhh1 antiserum and anti-rabbit (FITC) antibody (top panel) or only with anti-rabbit (FITC) antibody (lower panel). The right panel shows the corresponding DNA (DAPI) staining. (B) Purification of Dhh1-interacting proteins. Dhh1-interacting proteins were purified from Dhh1–ZZ-expressing cells by IgG affinity chromatography. Proteins were eluted using 1 M MgCl₂, separated on an 8% SDS gel and stained with Coomassie Blue. Proteins indicated by an arrow were identified by MALDI-TOF mass spectrometry. Thirty-six peptides out of 69 were matching peptides for Xrn1 (21% sequence coverage), 24 peptides out of 43 matched Pat1 (24% sequence coverage). Molecular weight markers are indicated on the left. The names of the proteins identified by MALDI are listed on the right. Xrn1* indicates a degradation product of Xrn1. Bands that are not specific for Dhh1p interaction are not labeled. As a control strain the isogenic wild-type background was used.

Fig. 2. Stabilization of CYH2 and STE2 mRNAs in dhh1Δ cells. Wild-type, dcp1Δ and dhh1Δ cells were grown to mid-log phase and transcription was inhibited using 1,10-phenanthroline. RNA was isolated at the indicated timepoints, separated by formaldehyde–agarose gel electrophoresis and analyzed by northern blotting. The decay of CYH2 mRNA (left panel) and STE2 mRNA (right panel) was examined. Numbers above the lanes indicate the time (in minutes) after transcriptional repression. RNA size markers are shown on the right.

Fig. 3. MFA2pG mRNA is stabilized in dhh1Δ cells. Wild-type and dhh1Δ strains carrying the GAL:MFA2pG reporter (Decker and Parker, 1993) were grown in 2% raffinose, 0.5% sucrose to an OD₆₀₀ of 0.6. Transcription of the reporter was induced for 8 min by the addition of 2% galactose and then repressed with 4% glucose. At the indicated time points (after transcriptional repression) cells were harvested and RNA was analyzed by northern blotting using an oligo(dC) probe. The arrows mark the full-length transcript as well as the decay intermediate.

Fig. 4. Transcriptional pulse–chase analysis of the PGK1pG reporter. Wild-type and dhh1Δ cells carrying the GAL:PGK1pG reporter (Decker and Parker, 1993) were grown to an OD₆₀₀ of 0.6. Transcription of the reporter was induced with 2% galactose for 8 min and then repressed with 4% glucose. At the indicated time points, cells were harvested and RNA was analyzed by northern blotting using an oligo(dC) probe. All samples were treated with RNase H to allow for the size resolution of the poly(A) tail of the reporter RNA on 6% polyacrylamide–urea gel electrophoresis. At time 0, the poly(A) tail of the RNA was also cleaved by RNAse H using an oligo(dT) as a marker for fully deadenylated mRNA (A0). The decay intermediate is marked with an arrow.

Fig. 5. Stabilization of capped mRNA species in dhh1Δ cells. RNA from wild-type, dcp1-2 (at the restrictive temperature) and dhh1Δ cells transformed with the MFA2pG reporter was isolated 0 and 15 min after transcription repression with galactose. RNA samples were subjected to treatment with purified Xrn1p in the presence (+) or absence (–) of EDTA to inhibit the exonuclease activity. RNA was separated by 1.2% formaldehyde–agarose gel electrophoresis and analyzed by northern blotting with a specific probe directed against the poly(G)-rich region in the reporter construct. Percentages indicate the ratio of capped, full-length transcripts versus total full-length transcripts calculated from four independent experiments. The sizes of the full-length transcripts as well as the decay intermediates, which serve as an internal control for Xrn1, are indicated by arrows. The lower panel represents a darker exposure of the wild-type, dcp1-2 and dhh1Δ panels at 15 min.

Fig. 6. Analysis of decapping activity of FLAG-Dcp1 in the presence of Dhh1. RNA was transcribed in vitro, capped and labeled using recombinant vaccinia capping enzyme in the presence of [α-³²P]GTP and S-adenosylmethionine. [α-³²P]GTP cap-labeled mRNA substrate (0.1 pmol) was used in the decapping reaction performed for 30 min at 37°C in the presence of FLAG-Dcp1. Reactions were terminated by addition of 0.5 M EDTA and analyzed by TLC using PEI cellulose plates. The results were visualized and quantified using a Phosphor Imager (Molecular Dynamics). Standards (Sigma) were developed on the TLC plates simultaneously; their migration is indicated on the right. (A) Lane 1 represents the input RNA. Resolution of nuclease P1 (Sigma)-treated substrate RNA is shown in lane 2. Migration of m⁷GDP released by 12.6 nM Dcp1 was followed in lane 3. An aliquot of the reaction in lane 3 was treated with NDPK (Sigma) (lane 4). (B) Decapping activity of FLAG-Dcp1 was titrated from 12.6 to 1.26 nM [lane 3 in (A) and lane 1 in (B), respectively]. Lane 2 shows decapping reactions performed with 1.26 nM FLAG-Dcp1 in the presence of equimolar recombinant Dhh1 purified from E.coli. The addition of 1.26 nM Dbp5 does not stimulate the release of m⁷GDP by Dcp1 (compare lanes 1 and 3). Dhh1p alone (lane 4) does not induce the release of m⁷GDP.