DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway - PubMed (original) (raw)

DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway

Erwin van Dijk et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown. In the 3'-5' mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP. In the 5'-3' pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5'-phosphorylated mRNA. We investigated mRNA decay in human cell extracts by using a new assay for decapping. We observed that 5'-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5'-3' mRNA decay pathway. Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated. Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms. The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human. m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay. m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay.

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Figures

Fig. 1.

Fig. 1.

Human HEK293 cell extract contains hDcp2-like decapping activity. (A) Outline of the RNA ligation assay used to detect decapped 5′ monophosphate RNA. In vitro transcribed, capped RNA (106 nt) is incubated with a source of decapping activity. The resulting 5′ monophosphate RNA is ligated to an RNA oligonucleotide. The ligation product is then reverse-transcribed (RT), and the resulting cDNA is PCR-amplified by using a primer complementary to the RNA oligonucleotide and a nested downstream primer. (B) Agarose gel analysis of PCR products. Decapping with hDcp2 (lane 1) and HEK293 whole cell extract (lane 2) is shown. Controls include heat-inactivated HEK293 whole cell extract (lane 3), reactions with HEK293 whole cell extract without substrate RNA (lane 4), and PCR without template (lane 5). A DNA size marker is in lane 6 with the fragment indicated on the right. The PCR product corresponding to full-length decapped substrate RNA is indicated by an arrow (left). Note that lane 2 contains faster migrating species in addition to the full-length product. An asterisk indicates nonspecific PCR products formed independently of the template and used as loading control. (C) Analysis of m7GDP formation with hDcp2 and human and yeast cell extract. Cap-labeled RNA was incubated with buffer (lane 1), 50 ng of GST-hDcp2-His-6 (24) (lane 2), 7 μg of human HEK293 whole cell extract (lane 3), or 7 μg of yeast BY4741 wild-type whole cell extract (lane 4). Reaction products were separated by TLC along with unlabeled standards that were revealed by fluorescence; their migration positions are indicated on the right. Pi, inorganic phosphate. An unidentified product X was formed upon incubation with yeast extract. The input RNA, which remained at the origin, is indicated by an arrow.

Fig. 2.

Fig. 2.

m7GDP conversion in human, yeast, and Xenopus cell extracts. (A) Radiolabeled m7GpppG was purified from TLC plates (Materials and Methods) and incubated with buffer (lane 2), HEK293 whole cell extract (lane 3), yeast BY4741 wild-type whole cell extract (lane 4), and whole cell extracts from Xenopus oocytes (lane 5). Lane 1 contains the input of the substrate without incubation. (B) As described for A, with radiolabeled m7GDP as substrate. (C) As described for A, with m7GMP as substrate. In all cases, 7 μg of cellular extract was used. Reaction products were separated by TLC. The migration positions of unlabeled standards are indicated on the sides. The unidentified product X resulting from incubation with yeast extract is also indicated.

Fig. 3.

Fig. 3.

m7GDP is stable in extracts from Δdcs1 cells. (A) Radiolabeled m7GpppG (lanes 1–5) or m7GDP (lanes 6–10) were incubated with buffer (lanes 2 and 7), BY4741 wild-type (WT) yeast extract (lanes 3 and 8), Δ_dcs2_ yeast extract (lanes 4 and 9), and Δ_dcs1_ yeast extract (lanes 5 and 10). Lanes 1 and 6 contain the input m7GpppG and m7GDP, respectively. Reaction products were separated by TLC; the migration positions of unlabeled standards are indicated on the right. Unidentified product X is also indicated. (B) m7GMP is converted to product X and free phosphate in all yeast extracts. Radiolabeled m7GMP was incubated with buffer (lane 2), BY4741 WT yeast extract (lane 3), Δ_dcs2_ yeast extract (lane 4), and Δ_dcs1_ yeast extract (lane 5). Lane 1 contains the input. Migration positions of m7GMP, product X, and free phosphate (Pi) are indicated.

Fig. 4.

Fig. 4.

Both yeast Dcs1p and human DcpS hydrolyze m7GDP to m7GMP. (A) Purified recombinant GST-DcpS-His-6 and GST-Dcs1-His-6 were fractionated by SDS/PAGE and detected by Coomassie staining. A protein size marker was run on the side. The two recombinant proteins have similar molecular mass (64 vs. 65 kDa, including the tags). (B) Radiolabeled m7GpppG (lanes 1–5) and m7GDP (lanes 6–10) were incubated with buffer (lanes 2 and 7), GST-hDcp2-His-6 (lanes 3 and 8), GST-DcpS-His-6 (lanes 4 and 9), and GST-Dcs1-His-6 (lanes 5 and 10). In all cases, 50 ng of protein was used. The inputs of m7GpppG and m7GDP are shown in lanes 1 and 6, respectively. Reaction products were separated by TLC; migration positions of unlabeled standards are indicated on the right. (C) m7GDP was incubated with buffer, GST-hDcp2-His-6, GST-DcpS-His-6, and GST-Dcs1-His-6 as in B; here, 150 ng of protein was used instead of 50 ng, and the incubation time was extended to1h(Materials and Methods).

Fig. 5.

Fig. 5.

Model showing the pathway-independent formation of m7GMP. After deadenylation, a mRNA can be degraded 3′–5′ by the exosome generating free m7GpppN. Alternatively, the mRNA can undergo decapping by hDcp2, generating m7GDP and a 5′-phosphorylated mRNA. RNAs degraded by the NSD and NMD pathways also generate m7GpppN and m7GDP, respectively. Both products are converted to m7GMP by DcpS. Thus, mRNA decay leads to the formation of m7GMP, irrespective of the pathway followed.

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