Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing.

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.

In eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.

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