ArabidopsisDCP2, DCP1, and VARICOSE Form a Decapping Complex Required for Postembryonic Development

Abstract

mRNA turnover in eukaryotes involves the removal of m 7 GDP from the 59 end. This decapping reaction is mediated by a protein complex well characterized in yeast and human but not in plants. The function of the decapping complex in the development of multicellular organisms is also poorly understood. Here, we show that Arabidopsis thaliana DCP2 can generate from capped mRNAs, m 7 GDP, and 59-phosphorylated mRNAs in vitro and that this decapping activity requires an active Nudix domain. DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS), an Arabidopsis homolog of human Hedls/Ge-1. Moreover, the interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. Consistent with their role in mRNA decay, DCP1, DCP2, and VCS colocalize in cytoplasmic foci, which are putative Arabidopsis processing bodies. Compared with the wild type, null mutants of DCP1, DCP2, and VCS accumulate capped mRNAs with a reduced degradation rate. These mutants also share a similar lethal phenotype at the seedling cotyledon stage, with disorganized veins, swollen root hairs, and altered epidermal cell morphology. We conclude that mRNA turnover mediated by the decapping complex is required for postembryonic development in Arabidopsis.

Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs

“…In animals, some of these complexes are associated with components of the RNA silencing machinery (Leung et al, 2006), and there is evidence that Drosophila P-bodies are involved in miRNA-directed target decay through deadenylation and decapping, whereas translational repression is P-body independent (Eulalio et al, 2007). In plants, components of the RNA decapping machinery were found involved in RNA silencing (Gazzani et al, 2004;Gy et al, 2007), and the existence of P-bodies was recently demonstrated (Xu et al, 2006). Thus, it will be interesting to investigate which type of proteins, such as other members of the large AGO family, are linked to miRNAs and their mRNA targets in nonpolysomal fractions.…”

The role of decapping proteins in the miRNA accumulation inArabidopsis thaliana

“…We selected heterozygotes and homozygotes of Arabidopsis mutant lines, dcp1-1 (GABI_844B03), 3 dcp1-2 (FLAG_563G05), 4 dcp2-1 (SALK_000519), 3,4 vcs-6 (SAIL_831_D08), 3 ago1-36 (SALK_087076), 23 se-1 (CS3176), 51 trs120-3 (SALK_111574), 42 hinkel (SAIL_1291_F04) and gnom (SALK_045424) by PCR-based genotyping. The primers used in the genotyping are listed in Table S1.…”

“…2A). 3,4,8 therefore, there was a possibility that the lethality caused the reduced levels of the miRNAs in the decapping mutants. To exclude this possibility, we examined the accumulation of the miRNAs in three other seedling lethal mutants (trs120, hinkel and gnom) by northern blotting.…”

“…[1][2][3][4][5] Previous studies using Arabidopsis thaliana have shown that a decapping complex is composed of Decapping 1 (DCP1), Decapping 2 (DCP2) and VARICOSE (VCS), a homolog of human Hedls/Ge-1. [2][3][4][6][7][8] DCP2 is the catalytic subunit. [9][10][11] The complex is mainly localized in cytoplasmic foci called processing bodies (P-bodies).…”

“…1,2,6 dcp1-, dcp2-and vcs-null mutants of A. thaliana show similar seedling lethal phenotypes, suggesting the importance of the decapping process in post-embryonic development. 3,4,8 Small RNAs of 20-24 nucleotide (nt) long are involved in gene silencing in eukaryotic gene regulation mechanisms. 12,13 The small RNAs are classified into two types; microRNAs (miRNAs) and short-interfering RNAs (siRNAs).…”