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

Abstract

3 ABSTRACT 49The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step 50 linking transcript synthesis and processing with translation. However, in plants, only few of 51 the factors that act in the mRNA export pathway have been functionally characterised. 52Flowering plant genomes encode several members of the ALY protein family, which function 53 as mRNA export factors in other organisms. Arabidopsis thaliana ALY1-4 are commonly 54 detected in root and leaf cells, but are differentially expressed in reproductive tissue. 55Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds 56 with higher affinity to ssRNA than dsRNA and ssDNA, and interacts preferentially with 5- THO rather than that of yeast (Yelina et al. 2010). Likewise, THO associates with UAP56, 99ALYs and MOS11 (the orthologue of CIP29) in Arabidopsis cells (Sørensen et al. 2017 (Germain et al. 2010;Lu et al. 2010;Pan et al. 2012;Xu et al. 2015; 114 Sørensen et al. 2017), but so far none of the ALY mRNA export adaptor candidates have been 115shown to function in nucleo-cytosolic transport of mRNAs in plants. 116In this study, we have systematically studied the Arabidopsis ALY proteins including their RESULTS 126 ALY proteins in Arabidopsis and other plants 127First, we compared the amino acid sequences of the four Arabidopsis ALY proteins (ALY1- sequence identity) as well as ALY3 and ALY4 (70% amino acid sequence identity) share a 137 high degree of sequence similarity, whereas the similarity of ALY1/2 versus ALY3/4 is 138 clearly lower (<42% amino acid sequence identity) (Supplemental Fig. S1B (Fig. 1A). ALY1 and truncated versions of the protein were expressed in 157 E. coli as 6xHis-GB1 fusion proteins, purified by two-step chromatography, and examined by 158 SDS-PAGE (Fig. 1B). For comparison we also used the unfused 6xHis-GB1 tag. The purified (Fig. 1C, Student's t-test, P < 0.05 and P < 0.001, respectively). The unfused 6xHis-GB1 165 tag did not exhibit affinity for ssRNA. To examine which domains of ALY1 contribute to the 166 RNA interactions, the binding to ssRNA of the different recombinant ALY1 versions was 167 measured (Fig. 1D (Fig. 3C, D). Therefore, the four ALY proteins and UAP56 228 are widely expressed in sporophytic cells of Arabidopsis plants. 229The subnuclear localisation of the ALY-GFP fusions was inspected in more detail by 230 CLSM in comparison to the UAP56-GFP and GFP-NLS controls. GFP-NLS (Antosch et al. immunofluorescence microscopy analysis (Kammel et al. 2013 (Table S1). In leaf cells, the nucleoplasmic distribution of 238 the ALY proteins appeared more heterogeneous than in root cells particularly for ALY4 that 239 partially localised to nucleoplasmic foci (Fig. 4B). Notably, the nucleolar enrichment of 253In view of the apparently ubiquitous expression of the four ALY-GFP proteins in 254 sporophytic cells, their occurrence was analysed in male and female gametophytes by CLSM. 255In mature pollen grains (Fig. 5A), the fluorescent signal of ALY1-...

Early response of barley embryos to low‐ and high‐dose gamma irradiation of seeds triggers changes in the transcriptional profile and an increase in hydrogen peroxide content in seedlings

“…The former transcript encodes the ortholog to the GINS complex protein in A. thaliana , which plays an essential role in the initiation of DNA replication. The latter is the ortholog to the THO complex subunit 4B in the same organism, being related to the control of plant development (Pfaff et al, ). In addition, the repression of HORVU3Hr1G109230 (Table ) is also intriguing.…”

“…The latter is the ortholog to the THO complex subunit 4B in the same organism, being related to the control of plant development (Pfaff et al, 2018). In addition, the repression of HORVU3Hr1G109230 (Table 2) is also intriguing.…”

The RNA Export Factor ALY1 Enables Genome-Wide RNA-Directed DNA Methylation

“…However, aly1 mutants did display a wavy/wrinkled leaf blade ( Figure 3A), which is accounted for by the overexpression of SDC (Figure 2A), which generates this phenotype when RdDM is lost (Henderson and Jacobsen, 2008). Furthermore, aly1 did not have the other phenotypic hallmarks associated with the loss of siRNA production (Supplemental Figure 5; Pfaff et al, 2018). Second, we used RNA gel-blot analysis to determine that TAS3 21-to 22-nucleotide siRNAs accumulated to the wild-type levels in the aly1 mutant ( Figure 3B), even though RdDM of this locus was defective in aly1 ( Figure 1C).…”

“…To pinpoint what aspect of RdDM is perturbed in aly1 mutants, we performed an RNA immunoprecipitation (RIP) using an ALY1pro: ALY1-GFP (green fluorescent protein) transgene (ALY1-GFP; Pfaff et al, 2018), which partially complemented the loss of RdDM in the aly1-1 mutant ( Figure 5A). We performed RIP-seq of ALY1-GFP in the aly1-1 background in four replicates and two mock immunoprecipitations (IPs; Supplemental Data Set 1).…”

“…Once genic mRNAs have been transcribed and processed, the TREX component RNA binding protein Yra1 (Aly/REF in metazoans, ALY in plants) is recruited to the mRNA to facilitate mRNA nuclear export (reviewed in Cullen, 2003). Four ALY proteins exist in Arabidopsis (ALY1 to ALY4; Pfaff et al, 2018). Proteins in this family contain RNA recognition motifs, and in metazoans and yeast their recruitment to mRNAs is dependent on interaction with the RNA helicase UAP56 (Sub2 in yeast; Luo et al, 2001;Strässer and Hurt, 2001).…”