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. 2003 May 2;300(5620):805-8.
doi: 10.1126/science.1082320.

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies

Ujwal Sheth  1 , Roy Parker
Affiliations

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies

Ujwal Sheth et al. Science. .

Abstract

A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.

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Figures

Fig. 1
Fig. 1
Decapping factors and Xrn1p localize to discrete foci in the cell. Proteins involved in mRNA decay were tagged with GFP following the PCR-based gene modification method described by Longtine et al. (17). (A) No GFP control. Cells expressing GFP tagged versions of factors involved in decapping are shown in (B to F) and factors involved in other aspects of mRNA turnover are shown in (H to L), and (G) shows untagged version of GFP only. The protein observed by GFP tagging is shown in the bottom left corner of each panel.
Fig. 2
Fig. 2
Alteration of mRNA decay affects the size and the number of P bodies. Using Dhh1p-GFP as a marker for P bodies, P bodies were observed in strains which are deleted for mRNA decay factors. (A) shows P bodies in WT cells, (B to F) show P bodies in dcp1Δ, xrn1Δ, ccr4Δ, pat1Δ, and lsm1Δ cells, respectively. Average number of P bodies in dcp1Δ and xrn1Δ strains per cell were 6.25 ± 3.7 and 12.7 ± 7.8, respectively, which, compared with wild-type strains in a Student's t test, gave P values of ≪0.001.
Fig. 3
Fig. 3
RNA decay intermediates are localized to P bodies. (A) WT (left column) and xrn1Δ (right column) cells are shown expressing the MFA2 mRNA with poly(G) and MS2 sites (top row), or the MFA2 mRNA with only a poly(G) tract (middle row), or the MFA2 mRNA with only the MS2 sites (bottom row). The schematic diagram of the reporter RNA expressed from the plasmid and its interaction with the MS2-GFP fusion protein is shown on the left. For simplicity, only a single protein is shown bound to a stem loop; however, because MS2 coat protein binds as a dimer, at least two MS2-GFP molecules are bound per site. (B) Left, RNA; middle, Lsm1p-RFP; and right, the merge generated by Adobe Photoshop. (C) WT cells expressing only the MS2-GFP fusion protein (left) or with a PGK1 reporter mRNA with a poly(G) tract and MS2 binding sites (right).
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References

    1. Shyu AB, Belasco JG, Greenberg ME. Genes Dev. 1991;5:221. - PubMed
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