The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors

Antosz, Wojciech; Pfab, Alexander; Ehrnsberger, Hans F.; Holzinger, Philipp; Köllen, Karin; Mortensen, Simon A.; Bruckmann, Astrid; Schubert, Thomas; Längst, Gernot; Griesenbeck, Joachim; Schubert, Veit; Grasser, Marion; Grasser, Klaus D.

doi:10.1105/tpc.16.00735

Cited by 125 publications

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“…According to standard protocol, Arabidopsis thaliana (Col-0) was grown at 21°C in a growth 530 chamber under long-day conditions (16h photoperiod per day) on soil (Antosz et al 2017), 531 while for some analyses plants were grown on MS medium (Murashige and Skoog 1962).…”

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

et al. 2018

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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-...

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

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“…These data argue that reduced histone acetylation within gene-bodies results from the process of RNAPII elongation, although different specificities of histone acetylation antibodies could also contribute to this observation. Proteomic screens identified several histone deacetylases (HDACs) associating RNAPII elongation complexes in Arabidopsis (Antosz et al, 2017). It remains to be tested which complexes provide HDAC activity linked to the suppression of intragenic TSSs.…”

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“…Many factors characterizing POINS are active in plants (Hajheidari et al, 2013;Van Lijsebettens and Grasser, 2014). The Arabidopsis FACT complex is physically associated with multiple RNAPII elongation factors, chromatin modifiers and elongation specific RNAPII isoforms (Antosz et al, 2017;Duroux et al, 2004). Reduced FACT activity results in developmental defects (Lolas et al, 2010) linked to imprinting (Ikeda et al, 2011), yet the role of FACT in TSS selection in plants is unclear.…”

Section: Introductionmentioning

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Transcription-driven Chromatin Repression of Intragenic Promoters

Nielsen

Ard

Leng

et al. 2018

Preprint

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Summary:Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a "positional information system" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The FACT histone chaperone complex is required for this repression mechanism. Genome-wide mapping of Transcription Start Sites (TSSs) reveals thousands of discrete intragenic TSS positions in FACT mutants. Histone 3 lysine 4 mono-methylation poises exonic sites to initiate RNAPII transcription in FACT mutants. Uncovering the mechanism for intragenic TSS repression through the act of RNAPII elongation has important implications for understanding pervasive RNAPII transcription and the regulation of transcript isoform diversity.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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“…But especially useful are isolated and flow-sorted cell nuclei free of disturbing cytoplasm (Dürr et al, 2014; Weisshart et al, 2016; Antosz et al, 2017) ( Figure 2 ).…”

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Super-resolution Microscopy – Applications in Plant Cell Research

Schubert

2017

Front. Plant Sci.

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Most of the present knowledge about cell organization and function is based on molecular and genetic methods as well as cytological investigations. While electron microscopy allows identifying cell substructures until a resolution of ∼1 nm, the resolution of fluorescence microscopy is restricted to ∼200 nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labeled structures and molecules. The recently developed super-resolution microscopy techniques, such as Structured Illumination Microscopy, Photoactivated Localization Microscopy, Stochastic Optical Reconstruction Microscopy, and Stimulated Emission Depletion microscopy allow analyzing structures and molecules beyond the diffraction limit of light. Recently, there is an increasing application of these techniques in cell biology. This review evaluates and summarizes especially the data achieved until now in analyzing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.

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The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors

Cited by 125 publications

References 87 publications

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

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

Transcription-driven Chromatin Repression of Intragenic Promoters

Super-resolution Microscopy – Applications in Plant Cell Research

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