The decapping enzyme Dcp1 participates in translation termination through its interaction with the release factor eRF3 in budding yeast

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Abstract

One of the rate-limiting steps in messenger RNA decay pathway is the 5′-cap cleavage of mRNAs, decapping reaction, which is conducted by the protein complex of Dcp1 and Dcp2. We find here that Dcp1p can interact with the release factor eRF3p (Sup35p) in Saccharomyces cerevisiae. Knockout of DCP1 caused not only the accumulation of nonsense mRNAs possibly due to the impaired decapping activity but also the enhancement of the read-through of nonsense codon. To examine the relationship between the two DCP1-knockout phenotypes, we produced DCP1 point mutants that lack the ability to support the translation termination. Interestingly, decapping activity of Dcp1p was still intact, but its interaction with eRF3p was abolished in the DCP1 mutants, indicating that the two functions originated from different entities of Dcp1p. These results suggest that the decapping enzyme Dcp1p may have an additional role in the translation termination through its interaction with eRF3p.

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Materials and methods

Yeast strains. All yeast strains used in this study are listed in Table 1. The yeast cells were grown in standard culture media and transformed with DNA by the lithium acetate method. Disruption of yeast genes was performed using PCR-based methods [9], and the disruption was confirmed by the phenotypic analysis and/or polymerase chain reactions (PCRs) with primers specific for the genes. Epitope tagging of yeast eRF3 (SUP35) was performed by the one-step method described by [10]. The

The decapping enzyme Dcp1p interacts with the eukaryotic release factor eRF3p/Sup35p

To elucidate the relationship between the decapping reaction and the translation termination reaction, we examined whether the decapping enzyme Dcp1p interacts with the release factor eRF3p in yeast. For the analysis, we introduced the plasmid expressing FLAG-tagged Dcp1p into wild-type cells whose chromosomal copy of SUP35 (encoding yeast eRF3) was tagged with Myc at its C terminus. As shown in Fig. 1, immunoprecipitation assay revealed that FLAG-Dcp1p interacts with eRF3p-Myc (the lower

Discussion

In S. cerevisiae, overexpression of both eRF1p and eRF3p enhances the translation termination at all three stop codons [15], indicating that the eRF1p–eRF3p complex acts as a key player at the ribosomal A site in the termination reaction. We previously reported that the GTP-bound form of eRF3p is required for the interaction with eRF1p to stimulate the termination reaction [3]. As shown in Fig. 5, eRF3 consists of an EF1α-like (GTP-binding) C-domain and a unique short N-domain [16]. The

Acknowledgments

This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government, Japan Society for the Promotion of Science (JSPS), the Mitsubishi Foundation, and the Uehara Memorial Foundation.

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      Citation Excerpt :

      eRF3 has been shown previously to mediate poly(A) shortening and mRNA decay in a manner coupled to translation termination [47]. Furthermore, yeast eRF3 directly interacts with the decapping protein Dcp1p [48], thus linking translation termination to decapping. Significantly, eRF3 and human PAN2/3 deadenylase compete for their binding to the poly(A) binding protein, PABPC1.

    1

    These authors contributed equally to this work.

    2

    Present address: Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan.

    3

    Present address: Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Nagoya 467-8603, Japan.

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