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. 2018 Nov 6;9:1602.
doi: 10.3389/fpls.2018.01602. eCollection 2018.

Functional Characterization of SMG7 Paralogs in Arabidopsis thaliana

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Free PMC article

Functional Characterization of SMG7 Paralogs in Arabidopsis thaliana

Claudio Capitao et al. Front Plant Sci. .
Free PMC article

Abstract

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7-like (SMG7L) paralogs in Arabidopsis thaliana. SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

Keywords: Arabidopsis; SMG7; gene duplication; meiosis; nonsense mediated RNA decay.

Figures

FIGURE 1
FIGURE 1
Comparison of Arabidopsis smg7-7 and smg7-1 mutants. (A) Schematic diagram of the SMG7 gene with the position of the smg7-7 mutation indicated. (B) Approximately 6-week-old wild type and mutant plants. (C) Anthers with viable pollen visualized by Alexander staining. (D) Developing pollen mother cells within an anther stained by DAPI. Tetrads are apparent in wild type, while late smg7 meiocytes contain randomly distributed chromatids. (E) Effect of smg7 alleles on the relative abundance of two alternatively spliced variants of the same transcript as determined by real time RT-PCR. Error bars represent SEM of three biological replicas. Asterisks indicate statistical significance of difference from wild type (P < 0.5, ∗∗P < 0.01, ∗∗∗P < 0.001, two-tailed t-test).
FIGURE 2
FIGURE 2
Double mutants of SMG7 and SMG7L. (A) Diagram of SMG7 and SMG7L genes with positions of T-DNA insertions and functional domains indicated. (B) qRT-PCR analysis of SMG7L and SMG7 transcripts. Positions of the regions amplified by PCR are indicated in (A). Error bars represent SEM of three biological replicas. (C) Five week-old wild type and mutant plants.
FIGURE 3
FIGURE 3
Fertility in plants deficient for SMG7L and SMG7. (A) Anthers with viable pollen visualized by Alexander staining. (B) Pollen count per anther in smg7-6 and smg7l-1 smg7-6. Error bars represent standard deviation from 35 anthers collected from 10 floral buds. (C) Proportion of siliques at a given position on the main stem producing at least one seed (1 represents the bottom-most silique). 9–20 plants were counted for each genotype.
FIGURE 4
FIGURE 4
Relative abundance of transcripts known to be targeted by NMD (Gloggnitzer et al., 2014). Error bars represent SEM from three biological replicas. Asterisks indicate statistical significance of difference from wild type (P < 0.5, ∗∗P < 0.01, ∗∗∗P < 0.001); numbers above horizontal bars represent significance of difference between SMG7L deficient plants and their respective controls.
FIGURE 5
FIGURE 5
Complementation of smg7-1 with SMG7K77ER185E. (A) Schematic diagram of the SMG7 gene with the K77E and R185E mutations indicated. (B) Five week-old T2 smg7-1 plants complemented with either the wild type or SMG7K77ER185E construct. (C) Anthers assayed by Alexander staining. (D) Relative abundance of SMG7 and two NMD reporter genes determined by real time RT-PCR. Two sets of primers were used for quantification of SMG7 mRNA (S1, S3) and their position is indicated in panel A. Error bars represent SEM from three biological replicas. Asterisks indicate statistical significance of difference from smg7-1 mutants (P < 0.5, ∗∗P < 0.01, ∗∗∗P < 0.001, two-tailed t-test). Plants derived from three independent transformants are shown in each category in (B–D).
FIGURE 6
FIGURE 6
Amino acid sequence motifs specific for the Arabidopsis SMG7 protein. (A) Schematic representation of the SMG7 protein and of its domains. Amino acid coordinates for domain boundaries, positions of SMG7-specific regions, and disruptions of SMG7 by T-DNA insertions are indicated. (B) Consensus sequences of individual SMG7-specific regions assembled from alignment of proteins from 13 plant species (Supplementary Figure S1). The x-axis represent the position in the A. thaliana SMG7 protein sequence. The y-axis labels represent the information content of the position in sequence (in bits).

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