The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4–RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4–RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.
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-...
We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.
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