Characterization of EMU, the Arabidopsis homolog of the yeast THO complex member HPR1

Abstract

Diverse and precise control is essential for eukaryotic gene expression. This is accomplished through the recruitment of a myriad of proteins to a nascent messenger RNA (mRNA) to mediate modifications, such as capping, splicing, 3'-end processing, and export. Despite being important for every cell, however, the mechanism by which the formation of diverse messenger ribonucleoprotein (mRNP) particles contributes to maintaining intricate systems in the multicellular organism remains incompletely defined. We identified and characterized a mutant gene named erecta mRNA under-expressed (emu) that leads to the defective mRNA accumulation of ERECTA, a developmental regulator in the model plant Arabidopsis thaliana. EMU encodes a protein homologous to a component of the THO complex that is required for the generation of functional mRNPs. Further analysis suggested that EMU is genetically associated with SERRATE, HYPONASTIC LEAVES1, and ARGONAUTE1, which are required for proper RNA maturation or action. Furthermore, mutations in another THO-related gene led to embryonic lethality. These findings support the presence and importance of the THO-related complex in plants as well as yeast and vertebrates.

FIGURE 1.

emu confers pleiotropic phenotypes. (A) RT-PCR analysis of ER expression in emu. RNA samples were prepared from shoots grown for 10 d after germination. The β-tubulin gene (TUB2) was amplified as a control. (B−D) Wild-type (WT) (B) and emu (C,D) seedlings. Plants were grown for 3 d after germination. (E−G) Wild-type (E) and emu (F,G) embryos. The same emu embryo was visualized at different foci to show three cotyledons (F,G). An asterisk indicates abnormal cell division. (H,I) Leaf series of wild-type (H) and emu (I) plants. For each genotype, the left two leaves are cotyledons, and the right-most leaf is the first cauline leaf. Plants were grown for 26 d after germination. (J−M) Wild-type (J), emu (K), er-105 (L), and emu er-105 (M) plants grown for 46 d after germination. emu er-105 double mutants exhibited a novel phenotype, which neither emu nor er confers. Arrows indicate the primary inflorescences. Photographs were taken at the same magnification. Scale bars: 1 mm in B−D; 20 μm in E−G; 1 cm in H,I.

FIGURE 2.

Map-based cloning of EMU. (A) Genetic mapping of EMU. The markers used for mapping are shown as well as the number of recombinants between emu and these markers. (B) Gene structure of EMU/At5g09860, with exons represented as closed boxes. The site of the lesion and nucleotide change in the emu allele is indicated. (C−E) Complementation of emu. Constitutive expression of wild-type At5g09860 cDNA in the emu mutant restored the wild-type appearance (D). Plants were grown for 17 d after germination. (F) EMU expression was analyzed by RT-PCR. Total RNA was extracted from seedlings grown for 7 d after germination. EF1αA4 was amplified as a control. Scale bars: 1 cm.

FIGURE 3.

attho2 mutations lead to embryonic lethality. (A,B) Mature embryos extracted from a plant heterozygous for the attho2-1 mutant allele, exhibiting either the wild-type (A) or mutant (B) phenotype. (C) A wild-type late-heart stage embryo. (D) An embryo segregating among wild-type late-heart stage embryos. (E,F) Embryos segregating among wild-type heart stage embryos. Asterisks denote aberrant cell divisions. Scale bars: 100 μm in A,B; 20 μm in C−F.

FIGURE 4.

Double mutants between emu and miRNA mutants exhibit novel phenotypes. (A−H) Plants grown for 21 d after germination. (I−P) Primary inflorescences of plants grown for 51 d after germination. (A,I) Wild type; (B,J) emu; (C,K) se-1; (D,L) emu se-1; (E,M) hyl1-2; (F,N) emu hyl1-2; (G,O) ago1-27; and (H,P) emu ago1-27. Scale bars: 1 cm.

FIGURE 5.

Alternative splicing patterns of SR genes are altered in emu. RT-PCR analysis of the expression of SR genes in seedlings grown for 7 d after germination. (A) Alternative splicing patterns of some SR genes are altered in the emu mutant. (B) Expression of EMU cDNA restored the wild-type expression pattern of RS31. Either CF701-60 (60), CF701-66 (66), or CF701-64 (64) contained a single copy of the 35S∷EMU transgene as a homozygote and was used for analysis.

FIGURE 6.

miRNA accumulation is reduced in emu. (A) RNA samples were prepared from inflorescence apices. (B) RNA samples were prepared from rosette leaves (left) and inflorescences (right) from plants grown for 27 d after germination. Ethidium bromide-stained gels on which rRNA and tRNA bands are visible are shown as a loading control. The intensity of the hybridization signal relative to WT is indicated and was calculated after normalization to rRNA.