Conserved RNaseII domain protein functions in cytoplasmic mRNA decay and suppresses Arabidopsis decapping mutant phenotypes

Abstract

Both transcription and RNA decay are critical for normal gene regulation. Arabidopsis mutants with defects in VARICOSE (VCS), a decapping complex scaffold protein, lack mRNA decapping and 5'-to-3' decay. These mutants show either severe or suppressed phenotypes, depending on the Arabidopsis accession. Here, we show that the molecular basis for this variation is the SUPPRESSOR OF VARICOSE (SOV), a locus that encodes a conserved, cytoplasmically localized RRP44-like RNaseII-domain protein. In vivo RNA decay assays suggest that active forms of this protein carry out decay on mRNA substrates that overlap with those of the decapping complex. Members of this conserved gene family encode proteins lacking the PIN domain, suggesting that SOV is not a functional component of the RNA exosome.

Fig. 1.

The L.er accession encodes a suppressor that modifies vcs mutant RNA accumulation defects. (A) The Col-0 and L.er accessions appear very similar, but vcs alleles isolated in these two genetic backgrounds have different phenotypes. In Col-0, the vcs-7 mutant is chlorotic and fails to make leaves, whereas the vcs-1 mutant, isolated in L.er, is green and produces broad leaves. (B) Both suppressed and severe vcs phenotypes are among the F₂ after crosses of vcs-1 and vcs-7 mutants to Col-0 and L. er, respectively. (C) The relative expression of four RNAs in 3-d seedlings of both wild-type accessions and in vcs-1 (L.er) and vcs-7 (Col-0), determined by using quantitative real-time RT-PCR. These mRNAs accumulate to high levels in vcs-7, and in each case, the RNA accumulates to a lower level in the vcs-1 mutant. Error bars indicate SE (s.e.m.). (D) RNA ligation assay for the presence of a 5′ cap. Tobacco acid pyrophosphatase (TAP) addition indicated by a (+). Note that PCR products are produced in vcs-7 and vcs-1 mutants only after incubation with TAP, indicating that the abundant RNAs quantified in C retain their 5′ cap. (Scale bars: 1 mm.)

Fig. 2.

Molecular characterization of the SUPPRESSOR OF VARICOSE. (A) The mapping interval that contained SOV included nine genes (depicted by broad arrows) and was defined by recombination break points indicated by the heavy vertical black arrows. (B) The L.er genomic DNA used for complementation analysis is represented by the dark gray bars, which were positioned to correspond to the region shown above the bars in A. Each rescue construct contained ≈2-kb upstream sequences, 1-kb downstream sequences, and all introns and exons. Note that the 5-kb scale bar works for both A and B. (C) Semiquantitative RT-PCR analysis of candidate gene expression in 7-d seedlings. Three of the candidate genes showed different levels of expression in the two accessions. Note that for At1g77690, reactions with different cycle numbers are shown to confirm differential expression. (D) Cartoon representation of the protein polymorphisms found in three SOV candidate genes. (Scale bar: 100 amino acids.)

Fig. 3.

Suppression of vcs is conferred by At1g77680 encoding a proline at position 705. (A) The effect of SOV genomic clones transformed into the wild-type and vcs-7 mutants; only SOV encoding PRO-705 confer the partially rescued vcs phenotype. Transgenic wild-type carrying SOV^L.er appear normal, and vcs-7 carrying SOV^L.er appear indistinguishable from vcs-1. This partially suppressed vcs-1 phenotype is mostly easily seen by the presence of leaves and green organ color. The partial suppression required the L.er version of SOV because vcs-7 transgenic plants carrying an extra copy of SOV^Col-0 were not suppressed unless SOV^Col-0 had been modified to resemble the L.er version at amino acid 705. (B) Rescue constructs designed to test the importance of PRO at position 705 and used in the experiments shown in A.

Fig. 4.

SOV encodes a conserved protein that localizes to the cytoplasm. (A) A phylogenetic tree, built using amino acid sequences of RNA binding domains (RNB) from SOV and SOV-like proteins. This analysis revealed that SOV as belonging to a conserved cohort that was distinct from RRP44 proteins. To the right, the domain organization of each analyzed protein is depicted. The RRP44 cohort all contained the typical RRP44 domains, whereas those of the SOV cohort all lacked the PIN domain and contained fewer CSD domains. Bootstrap values are indicated in blue. At, Arabidopsis; Sc, yeast; Mm, mouse; Ce, Caenorhabditis elegans; Dm, Drosophila; Os, rice; Sm, Selaginella. (B) Confocal image of CFP expression driven from the 35S promoter. CFP alone (Left) localizes throughout the cytoplasm and nucleus, whereas a translational fusion to SOV localizes to discrete cytoplasmic foci (Center) and a translational fusion to AtRRP44 largely localizes to the nucleus (Right). (Scale bars: 1 mm.)

Fig. 5.

SOV functions in cytoplasmic mRNA decay. (A) Steady-state levels of three mRNAs, relative to the actin internal control. (B) RNA decay analysis comparing three mRNAs in five genotypes: wild-type (Col-0), wild-type (L.er), vcs mutants (vcs-7 in Col-0 and vcs-1 in L.er) and transgenic vcs-7 (Col-0) containing SOV^L.er. (C) Relative expression of RRP44 and SOV in various organs of L.er plants. Both genes are expressed in all tissues analyzed; the relative abundance of SOV and AtRRP44 varied among the organs, but SOV expression was always greater than that of AtRRP44. All three analyzes used real-time RT-PCR. Error bars indicate SEM.