ALY RNA-Binding Proteins Are Required for Nucleocytosolic mRNA Transport and Modulate Plant Growth and Development

Abstract

The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only a few of the factors that act in the mRNA export pathway have been functionally characterized. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis (Arabidopsis thaliana) ALY1 to ALY4 are commonly detected in root and leaf cells, but they are differentially expressed in reproductive tissue. Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds with higher affinity to single-stranded RNA than double-stranded RNA and single-stranded DNA and interacts preferentially with 5-methylcytosine-modified single-stranded RNA. Compared with the full-length protein, the individual RNA recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 complements the lethal phenotype of yeast cells lacking the ALY1 ortholog Yra1. Whereas individual aly mutants have a wild-type appearance, disruption of ALY1 to ALY4 in 4xaly plants causes vegetative and reproductive defects, including strongly reduced growth, altered flower morphology, as well as abnormal ovules and female gametophytes, causing reduced seed production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our results highlight the requirement of efficient mRNA nucleocytosolic transport for proper plant growth and development and indicate that ALY1 to ALY4 act partly redundantly in this process; however, differences in expression and subnuclear localization suggest distinct functions.

Figure 1.

Production of recombinant ALY1 proteins and MST analysis of their interactions with nucleic acids. A, Schematic representation of ALY1 depicting the different domains of the protein. The RRM and the relatively variable N- and C-terminal domains (compare with Supplemental Fig. S1) are indicated. Numbers indicate amino acid sequence positions that delimit the domains. B, SDS-PAGE analysis of purified full-length and truncated ALY1 expressed in E. coli, alongside the unfused 6xHis-GB1 tag. C, MST analysis of the interaction of full-length ALY1 with different nucleic acids. Incubation of the unfused 6xHis-GB1 tag with ssRNA was included as a control. D, MST analysis of the interaction of full-length and truncated versions of ALY1 with ssRNA. E, MST analysis of the interaction of full-length ALY1 with m⁵C-modified 19-nucleotide ssRNA and with the unmodified control RNA. Each data point represents two biological and two technical replicates, and error bars indicate sd of the biological replicates.

Figure 2.

ALY proteins interact with UAP56 in yeast two-hybrid assays and FRET analyses. A, Yeast two-hybrid assays with cells harboring the indicated constructs grown on DDO (left) or QDO (right) medium. B, Protein interactions analyzed by FRET-APB. N. benthamiana leaves were coinfiltrated with vectors expressing donor (eGFP, ALY1, and ALY3; green)/acceptor (mCherry and UAP56; red) combinations as indicated, alongside positive and negative controls. Transiently transformed cells were analyzed using two biological replicates by FRET-APB. Mean FRET-APB efficiencies (±sd, eight analyzed nuclei each) are shown. ***, Statistically significant difference by Student’s t test (P < 0.001).

Figure 3.

Localization of ALY1- to ALY4-GFP and UAP56-GFP fusion proteins in nuclei of root and leaf cells. A, Root tips of plants at 8 d after stratification (DAS) expressing the indicated GFP fusion proteins under the control of the respective native promotors. GFP fluorescence is visible in green, while propidium iodide staining is in red. B, Root tips as in A shown at higher magnification. C, Abaxial surfaces of the second leaf pair from 14-DAS plants depicting GFP fluorescence in nuclei of epidermal cells and of stomatal guard cells. D, Magnified images of guard cells with nuclear GFP fluorescence. Bars = 50 µm (A and B), 25 µm (C), and 10 µm (D).

Figure 4.

Subnuclear localization of ALY1- to ALY4-GFP and UAP56-GFP fusion proteins in nuclei of root and leaf cells. A, Root cell nuclei from 8-DAS plants expressing the indicated GFP fusion proteins under the control of the respective ALY native promotors. The images depicted represent the most commonly observed subnuclear distributions of fusion protein. B, Cell nuclei from the second leaf pair of 14-DAS plants of the same GFP fusion protein lines depicted in A. n, Nucleoli. Bars = 5 μm.

Figure 5.

Localization of ALY-GFP fusion proteins in pollen grains and ovules. A, Mature pollen grains. Closed and open arrowheads indicate sperm cell nuclei and vegetative nuclei, respectively. DNA is counterstained with DAPI (blue). GFP fluorescence (left images) and the corresponding bright-field images, merged with fluorescent signals (right images), are shown. B, Mature unfertilized ovules. GFP fluorescence (left images) and the corresponding bright-field images, merged with GFP signals (right images), are shown. Insets depict ALY-GFP signals in the nuclei of female gametophytic cells. Closed and open arrowheads and asterisks indicate synergid nuclei, egg cell nuclei, and central cell nuclei, respectively. Bars = 5 µm (A) and 25 µm (B).

Figure 6.

Phenotype of the quadruple aly mutant plants (4xaly) relative to Col-0. A and B, Representative images of plants at 28 DAS (A) and 42 DAS (B). Plants were grown under long-day conditions in soil. C, Roots of 8-DAS plants grown on solid Murashige and Skoog (MS) medium.

Figure 7.

4xaly plants produce a number of flowers with altered morphology and abnormal ovules. A to F, Flower morphology. A, Col-0 flower depicting normal petal number. B, 4xaly flower depicting altered petal number. C, Normal 4xaly flower as observed in the majority of cases. D, Col-0 flower depicting normal trichome number on sepals and stamen filament length (closed arrowheads). E and F, Abnormal 4xaly flower depicting increased trichome number on sepals (open arrowheads) and reduced stamen filament length (closed arrowheads). G to K, Differential contrast microscopy of cleared ovules. G, Col-0 ovule depicting normal morphology. H, 4xaly ovule depicting normal morphology. I, 4xaly ovule depicting arrested female gametophyte development. J, 4xaly ovule depicting collapsed female gametophyte. K, 4xaly ovule depicting abnormal integument development. Closed and open arrowheads and asterisks indicate synergid cell nuclei, egg cell nuclei, and central cell nuclei, respectively. FG, Female gametophyte; II, inner integuments; OI, outer integuments. L and M, Morphology of self-pollinated siliques of the indicated genotypes (pistil × pollen). N to Q, Morphology of cross-pollinated siliques of the indicated genotypes (pistil × pollen). Open arrowheads in M indicate gaps with undeveloped seeds in a 4xaly silique. Bars = 0.2 mm (A–F), 20 µm (G–K), and 2 mm (L–Q).

Figure 8.

Complementation of the yeast yra1 mutant and analysis of the nucleocytosolic distribution of polyadenylated mRNA in 4xaly plants. A, Growth of the YRA1 shuffle strain (yra1::HIS3, pURA3-Yra1) following complementation with Arabidopsis ALY1 or yeast YRA1 alongside an empty-vector negative control. For ALY1, three individual transformants were analyzed. Cells were grown on 5-FOA plates, thus only allowing growth of yra1::HIS3 cells that have lost the pURA3-Yra1 plasmid. The plate was photographed after 18 d at 24°C to better visualize small, slow-growing colonies. B, Root cells of 6-DAS Col-0, 4xaly, and thp1 seedlings examined using whole-mount in situ hybridization with a fluorescently labeled 48-nucleotide oligo(dT) probe. CLSM images (taken using identical microscope settings) of representative regions of the analyzed roots (top row) and individual cells (bottom row) are shown. Bars = 60 μm (top row) and 10 μm (bottom row). C, The fluorescent hybridization signal of nuclei relative to cytosol as quantified for 58 or more nuclei from three roots of each genotype. The ratios are depicted relative to Col-0 (ratio of 1), with error bars indicating sd. ***, Statistically significant difference by two-tailed Student’s t test (P < 0.001).