ALY RNA-binding proteins are required for nucleo-cytosolic ... · 3/14/2018 · 3 49 ABSTRACT 50 The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical

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ALY RNA-binding proteins are required for nucleo-cytosolic mRNA transport and 6

modulate plant growth and development1 7

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Christina Pfaff a, Hans F. Ehrnsberger

a, María Flores-Tornero

a, Brian B. Sørensen

a, Thomas 12

Schubertb, Gernot Längst

b, Joachim Griesenbeck

b, Stefanie Sprunck

a, Marion Grasser

a,2 and 13

Klaus D. Grassera,2

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a Department of Cell Biology & Plant Biochemistry 17

b Department for Biochemistry III 18

Biochemistry Centre, University of Regensburg, 19

Universitätsstr. 31, D-93053 Regensburg, Germany 20

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Short title: ALY mRNA export factors 23

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One sentence summary: Arabidopsis expresses four ALY family nuclear RNA-binding 26

proteins that interact with the RNA helicase UAP56 and their depletion causes nuclear mRNA 27

accumulation and developmental defects. 28

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Plant Physiology Preview. Published on March 14, 2018, as DOI:10.1104/pp.18.00173

Copyright 2018 by the American Society of Plant Biologists

www.plantphysiol.orgon October 19, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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Footnotes: 31

1 This work was supported by the German Research Foundation (DFG) through grant SFB960 32

to K.D.G. 33

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2 To whom correspondence should be addressed: Department of Cell Biology & Plant 35

Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, D-93053 36

Regensburg, Germany, Tel.: +49-941-9433033; Fax: +49-941-9433352; E-mail 37

[email protected], or Tel.: +49-941-9433032; Fax: +49-941-9433352; E-mail: 38

[email protected] 39

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The author responsible for distribution of materials integral to the findings presented in this 41

article in accordance with the policy described in the Instructions for Authors 42

(www.plantphysiol.org) is: Klaus Grasser ([email protected]). 43

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M.G. and K.D.G. conceived the project; G.L., J.G., S.S., M.G. and K.D.G. supervised the 45

project; C.P., H.F.E., M.F.-T., B.B.S., S.S., T.S. and M.G. conducted experiments; C.P., 46

H.F.E., M.F.-T., B.B.S., T.S. G.L., J.G., S.S., M.G. and K.D.G. analysed the data; S.S., M.G. 47

and K.D.G. wrote the article; all authors read and approved the final manuscript.48


mailto:[email protected]




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ABSTRACT 49

The 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. 52

Flowering 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. 55

Moreover, 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 5-57

methylcytosine-modified ssRNA. Compared to the full-length protein, the individual RNA 58

recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA 59

helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 60

complements the lethal phenotype of yeast cells lacking the ALY1 orthologue Yra1. Whereas 61

individual aly mutants have a wild-type appearance, disruption of ALY1–4 in 4xaly plants 62

causes vegetative and reproductive defects, including strongly reduced growth, altered flower 63

morphology, as well as abnormal ovules and female gametophytes, causing reduced seed 64

production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our 65

results highlight the requirement of efficient mRNA nucleo-cytosolic transport for proper 66

plant growth and development and indicate that ALY1–4 act partly redundantly in this 67

process; however, differences in expression and subnuclear localisation suggest distinct 68

functions. 69

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INTRODUCTION 71

In eukaryotic cells, pre-mRNAs are synthesised in the nucleus by RNA polymerase II and co-72

transcriptionally processed by diverse events including 5′ capping, splicing and 3′ 73

polyadenylation. Only when these processes are completed, the mature mRNAs are exported 74

from the nucleus to the cytosol for translation. In yeast and metazoa, a plethora of proteins 75

have been identified that mediate the transport of export-competent mRNAs across the 76

nuclear envelope through nuclear pore complexes (Köhler and Hurt 2007; Wickramasinghe 77

and Laskey 2015). The export of mRNAs is an integrated step in mRNA biogenesis, and the 78

TREX (TRanscription-EXport) multiprotein complex plays a central role in the coupling of 79

transcription, processing and export (Katahira 2012; Heath et al. 2016). TREX consists of the 80

THO core complex that associates with various other components including the RNA helicase 81

UAP56 (also known as DDX39B, Sub2 in yeast) and mRNA export factors such as 82

CIP29/SARNP (Tho1 in yeast) and ALY (Yra1 in yeast) (Sträßer et al. 2002; Masuda et al. 83

2005; Dufu et al. 2010). In metazoa, TREX is recruited to mRNAs in a capping- and splicing-84

dependent manner (Katahira 2012; Heath et al. 2016). In addition to its function in splicing, 85

UAP56 serves an important role in initiating the export of mRNAs by loading mRNA export 86

adaptors such as ALY (Ally of AML-1 and LEF-1 (Bruhn et al. 1997), also termed REF) onto 87

both spliced and intronless mRNAs (Luo et al. 2001; Taniguchi and Ohno 2008). mRNA 88

adaptors are recruited to the RNA molecule co-transcriptionally through various processing 89

events to licence mRNA for export (Walsh et al. 2010). ALY (and other TREX components) 90

recruit the mRNA export receptor NXF1/NXT1 (Mex67/Mtr2 in yeast) and hand the mRNA 91

over to NXF1/NXT1 (Viphakone et al. 2012). Finally, NXF1/NTF1 interacts with TREX-2 at 92

the nuclear pore and directs the mRNA through the nuclear pore by binding to FG repeats of 93

nucleoporins (Katahira 2012; Wickramasinghe and Laskey 2015; Heath et al. 2016). 94

In plants, only few factors that are involved in the above-mentioned mRNA export 95

pathway have been functionally characterised to date (Xu and Meier 2008; Merkle 2011; 96

Gaouar and Germain 2013). The composition of the Arabidopsis THO core complex of TREX 97

(consisting of HPR1, THO2, THO5A/B, THO6, THO7A/B and TEX1) resembles metazoan 98

THO rather than that of yeast (Yelina et al. 2010). Likewise, THO associates with UAP56, 99

ALYs and MOS11 (the orthologue of CIP29) in Arabidopsis cells (Sørensen et al. 2017). 100

MOS11 and ALY2 interact directly with the ATP-dependent RNA helicase UAP56 (Kammel 101

et al. 2013). Four putative mRNA export adaptors of the ALY family (ALY1–4) are encoded 102

by the Arabidopsis genome and in transient assays it was observed that they display a distinct 103



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subnuclear distribution (Uhrig et al. 2004; Pendle et al. 2005). They were identified as 104

interactors of the Tomato Bushy Stunt Virus (TBSV) P19 protein and the association with 105

P19 resulted in a relocation of ALY2 and ALY4 from the nucleus to the cytosol (Uhrig et al. 106

2004; Canto et al. 2006). Surprisingly, some ALY proteins were identified along with 107

components of the exon junction complex (EJC) in Arabidopsis nucleoli (Uhrig et al. 2004; 108

Pendle et al. 2005). ALY4 (and a closely related orthologue from Nicotiana benthamiana) 109

was reported to be involved in a variety of plant responses upon pathogen infection including 110

stomatal closure (Teng et al. 2014). 111

THO and TREX-2 subunits as well as MOS11 were experimentally demonstrated to be 112

involved in plant mRNA export, as mos11, hpr1, tex1 mos11 and thp1mutants exhibit nuclear 113

mRNA accumulation (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 115

shown to function in nucleo-cytosolic transport of mRNAs in plants. 116

In this study, we have systematically studied the Arabidopsis ALY proteins including their 117

expression and subcellular localisation. Moreover, we examined their interactions with RNA 118

and the RNA helicase UAP56. Mutant plants deficient in expression of all four ALY genes 119

exhibit nuclear mRNA accumulation and show various developmental defects both in the 120

sporophyte and the female gametophyte. Our results indicate that the Arabidopsis ALY1–4 121

play a role in mRNA export and that efficient nucleo-cytosolic transport of mRNAs is a 122

requirement for proper plant growth and development. 123

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RESULTS 126

ALY proteins in Arabidopsis and other plants 127

First, we compared the amino acid sequences of the four Arabidopsis ALY proteins (ALY1–128

4) as well as of putative orthologues from several other plants. The Arabidopsis ALY proteins 129

(245–295 amino acid residues; 25.8–31.3 kDa) are generally rich in the amino acid residues 130

arginine and lysine, and accordingly they are basic proteins with theoretical pI values of 9.8–131

11.6. An alignment of the amino acid sequences revealed that the four Arabidopsis ALY 132

proteins (like mammalian ALY proteins (Walsh et al. 2010)) have in common a centrally 133

positioned conserved RNA recognition motif (RRM) (Burd and Dreyfuss 1994) including 134

characteristic RNP sequences (Supplemental Fig. S1A). The RRM is flanked by more 135

variable Arg/Gly-rich N- and C-terminal domains. ALY1 and ALY2 (54% amino acid 136



6

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). Arabidopsis 139

ALY1/2 and ALY3/4 show ~44% and ~38% amino acid sequence identity with human ALY, 140

respectively (Fig. S1B). Searching protein sequences of several other plant model species 141

using Arabidopsis ALY1 as a query demonstrated that ALY-like proteins are present in 142

flowering plants (monocots, dicots, Amborella) as well as in Selaginella and Physcomitrella. 143

Monocot and dicot plants generally encode several ALY proteins (4 or 5 different genes), 144

whereas there seem to be only two ALY-like proteins in Amborella and one in Selaginella. A 145

multiple sequence alignment of these sequences revealed that the ALY amino acid sequences 146

of other flowering plants are similarly diversified as those of Arabidopsis (Supplemental Fig. 147

S2). 148

149

RNA-binding of ALY1 150

A multitude of proteins interacting with RNA have been identified in Arabidopsis (Köster et 151

al. 2017) and a critical feature of mRNA export adaptors is their ability to bind RNA (Walsh 152

et al. 2010). However, RNA interactions of plant ALY proteins have not been characterised 153

thus far and we selected the ALY1 protein for RNA-binding analyses. In addition to full-154

length ALY1 (aa1–245), we examined the predicted RRM (ALY1RRM, aa85–164) and 155

ALY1 lacking either the N-terminal domain (ALY1ΔN, aa85–245) or the C-terminal domain 156

(ALY1ΔC, aa1–164) (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 159

recombinant proteins were analysed for RNA-binding in solution using microscale 160

thermophoresis (MST). First, binding affinities were measured comparatively for 161

fluorescently labelled 25-nt ssRNA, dsRNA and ssDNA probes by incubation with increasing 162

concentrations of ALY1. Compared to dsRNA and ssDNA (KD = 918 201 and 1300 479 163

nM, respectively), ALY1 exhibited a preference for interaction with ssRNA (KD = 481 109 164

nM) (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, Student’s t-test, P < 0.005). Full-length ALY1 bound the RNA probe with 168

more than 5-fold higher affinity compared to that of the individual RRM (KD 414 99 vs. 169



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2285 315 nM). For the ALY1ΔN and ALY1ΔC proteins, intermediate affinities (KD 1700 170

741 and 830 388 nM, respectively) were determined. Therefore, for efficient RNA-binding 171

of ALY1, the RRM and the terminal domains are required and the contribution of the N-172

terminal domain seems to be greater than that of the C-terminal domain. Recently, it was 173

reported that 5-methylcytosine (m5C) is a common and important post-transcriptional 174

modification of mRNAs and other RNAs in Arabidopsis (David et al. 2017) and that 175

mammalian ALY preferentially binds to m5C-modified RNA (Yang et al. 2017). Therefore, 176

we comparatively analysed the interaction of Arabidopsis ALY1 with an m5C-modified 19-nt 177

ssRNA oligonucleotide relative to the unmodified control RNA. The MST measurements 178

demonstrated that ALY1 bound the m5C-modified RNA (KD = 77 32 nM) with about 3-fold 179

higher affinity than that for the unmodified RNA (KD = 249 88 nM; Fig. 1E, Student’s t-180

test, P < 0.01). The ALY proteins interact directly with the RNA helicase UAP56 181

The ALY proteins co-purify with the THO/TREX components TEX1 and UAP56 from 182

Arabidopsis cells (Sørensen et al. 2017) and ALY2 interacts with the RNA helicase UAP56 in 183

yeast cells and in vitro (Kammel et al. 2013). We examined whether all four ALY proteins 184

can interact directly with UAP56 using the yeast two-hybrid assay. UAP56 fused to the DNA-185

binding domain (BD) of GAL4 was expressed in yeast cells, whereas the ALY sequences 186

were fused to the activation domain (AD) of GAL4. Yeast cells co-expressing both types of 187

fusion proteins were scored for their growth. Whereas all strains grew on the double drop-out 188

(DDO) medium, only the strains expressing both the UAP56-BD and the AD-ALY fusion 189

proteins (along with a positive control) grew on the high-stringency, selective quadruple drop-190

out (QDO) medium (Fig. 2A), demonstrating that ALY1–4 interact with UAP56 in yeast 191

cells. Strains expressing only one of the two fusion proteins and the negative control showed 192

no growth on the QDO medium. The interaction between ALY1 and ALY3 with UAP56 was 193

further analysed by Förster resonance energy transfer (FRET) in Agrobacterium-infiltrated 194

tobacco leaves, introducing plasmids that drive the expression of eGFP and mCherry fusion 195

proteins as donor/acceptor pairs. The recovery of donor fluorescence after acceptor 196

photobleaching (APB) was quantified (Fig. 2B). An eGFP-mCherry fusion protein (27.9 197

1.3% FRET-APB efficiency) served as positive control for the experiment, while cells 198

expressing ALY1/3-eGFP along with unfused mCherry provided background levels (0.2 199

3.7%/0.7 3.5% FRET-APB efficiency). FRET-APB efficiencies of 11.4 3.6% and 9.3 200

1.8% were recorded for the combinations ALY1-eGFP/UAP56-mCherry and ALY3-201



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eGFP/UAP56-mCherry, respectively. Therefore, according to yeast two-hybrid assays and 202

FRET-APB analyses, ALY proteins interact directly with UAP56. 203

204

Expression and subcellular localisation of ALY proteins 205

The subnuclear distribution of ALY1–4-GFP fusion proteins was analysed previously in 206

infiltrated N. benthamiana leaves (Uhrig et al. 2004) and transiently transformed suspension 207

cultured Arabidopsis cells (Pendle et al. 2005). Since we intended to analyse the expression 208

and subcellular localisation of ALY-GFP fusion proteins in stably transformed plants, ideally 209

in the absence of the respective endogenous protein, we started out characterising T-DNA 210

insertion lines of the four ALY genes. According to PCR-based genotyping and sequencing of 211

the corresponding genomic regions, plants homozygous for T-DNA insertions within exon 212

sequences were obtained for each gene (Supplemental Fig. S3A, B). RT-PCR analyses 213

revealed that the respective ALY transcript was not detectable in the mutant plants, while the 214

level of the other ALY transcripts appeared unaltered (Supplemental Fig. S3C). The four aly 215

mutant lines had essentially a wild-type appearance (see below for details), and were 216

transformed with constructs driving the expression of the respective ALY-GFP fusion protein 217

under control of its promoter. Based on RT-PCR analysis of the different ALY-GFP 218

expressing plants, the ALY transcript levels in the transformed plant lines were comparable to 219

those in Col-0 (Supplemental Fig. S4). Root tips of the plants expressing ALY-GFP fusion 220

proteins (and for comparison UAP56-GFP) were stained with propidium iodide and analysed 221

by confocal laser scanning microscopy (CLSM). In agreement with immunofluorescence 222

analyses (Kammel et al. 2013), UAP56-GFP was detected exclusively in the cell nuclei (Fig. 223

3A, B). The GFP signal of the ALY proteins was also restricted to the cell nuclei, which is in 224

line with previous studies (Uhrig et al. 2004; Pendle et al. 2005). Expression of the four ALY 225

proteins and of UAP56 is detected in all root cells. Examination of leaf surfaces of these 226

plants revealed that the ALY-GFP and UAP56-GFP fluorescence was observed in the nuclei 227

of epidermal cells and guard cells (Fig. 3C, D). Therefore, the four ALY proteins and UAP56 228

are widely expressed in sporophytic cells of Arabidopsis plants. 229

The 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. 231

2015) was evenly distributed throughout root nuclei and UAP56 occurs in the nucleoplasm 232

with no clear signal in nucleoli (Fig. 4A), in agreement with the findings of an 233

immunofluorescence microscopy analysis (Kammel et al. 2013). In comparison, the 234



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subnuclear localisation of the ALY proteins is more variable, generally showing a less 235

uniform distribution in the nucleoplasm. Strikingly, in many root cells ALY3 and ALY4 were 236

strongly enriched in nucleoli, whereas ALY1 and ALY2 were clearly less prominent in 237

nucleoli relative to the nucleoplasm (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 240

ALY3 and ALY4 seen in root cells was not observed in leaf cells and the proteins were 241

similarly distributed in nucleoplasm and nucleoli. Like in root cells, ALY1 and ALY2 were 242

primarily detected in the nucleoplasm of leaf cells and less in nucleoli. Thus, in accordance 243

with their amino acid sequence similarities (Supplemental Fig. S1), the subnuclear 244

distribution of ALY1/2 differs to some extent from that of ALY3/4. Recently, the motif 245

WxHD was identified to play a role in the subnuclear localisation of mammalian ALY 246

(Gromadzka et al. 2016). Since this motif also occurs in ALY1 and a similar WxxD motif is 247

found in ALY2, but not in ALY3 and ALY4 (Supplemental Fig. S1), we considered whether 248

this motif might cause the distinct subnuclear localisation of Arabidopsis ALY proteins. 249

However, the analysis of plants expressing ALY1 and ALY3 versions with the WxxD motif 250

swapped between the two proteins (Supplemental Fig. 5A) did not reveal an altered 251

subnuclear distribution of these proteins when compared with control proteins (supplemental 252

Fig. 5B). 253

In 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. 255

In mature pollen grains (Fig. 5A), the fluorescent signal of ALY1-GFP and ALY2-GFP was 256

clearly visible in the vegetative nucleus. ALY2-GFP fluorescence was readily detectable in 257

the sperm cells, whereas the sperm cell-derived signal of ALY1-GFP was much weaker. 258

ALY3-GFP fluorescence was present in the vegetative nucleus but not detectable in the sperm 259

cells. Nevertheless, the lack of ALY3-GFP detection in sperm cells by CLSM may be due to 260

the overall weaker fluorescence of ALY3-GFP. By contrast, no fluorescence was detectable in 261

transgenic ALY4-GFP mature pollen grains, even when the pollen grains were not 262

counterstained with DAPI to exclude the possibility of DAPI-induced attenuated GFP 263

fluorescence. Comparable results were obtained after in vitro pollen germination, when the 264

fluorescence of the ALY-GFP fusion proteins was monitored in growing pollen tubes 265

(Supplemental Fig. S6). Detailed analysis of mature unfertilised ovules (Fig. 5B) 266

demonstrated that ALY1-GFP was detected specifically within the female gametophyte. The 267



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nuclei of the synergid cells and the homodiploid central cell nucleus showed strongest ALY1-268

GFP signals, whereas the fluorescence was much weaker in the egg cell nucleus. No 269

fluorescence was detected in the sporophytic tissues of ALY1-GFP-expressing ovules. 270

Compared with ALY1-GFP, ALY2-GFP and ALY4-GFP displayed similar expression 271

patterns in the nuclei of female gametophytic cells, with overall weaker fluorescence for 272

ALY4-GFP. By contrast, ALY3-GFP was expressed in the central cell nucleus and the egg 273

cell nucleus but was not detectable in the synergid cells. Furthermore, ALY2-GFP, ALY3-274

GFP and ALY4-GFP were also expressed in the sporophytic cells of the ovule such as the 275

inner and outer integuments, the nucellus and the funiculus. Therefore, in contrast to root and 276

leaf cells, the analysis of pollen grains and ovules established clear differences in the 277

expression pattern and the expression strength of the four ALY proteins. Moreover, unlike in 278

root cells, we did not observe any nucleolar enrichment of ALY3-GFP and ALY4-GFP in the 279

cells of the female gametophyte. 280

281

Plants lacking ALY expression exhibit various developmental defects 282

As mentioned above, plants of the four individual aly mutant lines are phenotypically 283

essentially unaffected. Therefore, we generated double mutants combining the single-284

mutations of the more closely related aly1 and aly2 as well as aly3 and aly4. Like single aly 285

mutants, the aly1 aly2 and aly3 aly4 double-mutant plants displayed basically a wild-type 286

appearance (Supplemental Fig. S7A), including normal flower architecture (Supplemental 287

Fig. S7B) and unaffected fertility (Supplemental Fig. S7C). The only noteworthy variance 288

was that the aly3 mutant (and consistently the aly3 aly4 double-mutant) bolted slightly earlier 289

than Col-0 (Supplemental Fig. S7A). In view of the very mild impairment of these plants 290

under standard growth conditions, we subsequently crossed the aly1 aly2 and aly3 aly4 291

double mutants. Plants homozygous for all four T-DNA insertions were obtained and these 292

aly1 aly2 aly3 aly4 quadruple mutants are herein referred to as 4xaly. The quadruple mutant 293

plants were phenotypically severely affected, as evident from the reduced size of leaves and 294

rosette diameter as well as from decreased number of primary inflorescences (Fig. 6A,B; 295

Supplemental Fig. S8A,B). At seedling stage, the hypocotyls of 4xaly plants were remarkably 296

elongated relative to that of Col-0 (Supplemental Fig. S8C). The mutant plants had fewer 297

leaves than Col-0 when they started bolting (Supplemental Fig. S8D), and the bolting of 4xaly 298

plants was delayed when compared with that of Col-0 (Supplemental Fig. S8E). In addition to 299



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the defects in the aerial parts, the growth of the primary root was clearly decreased (Fig. 6C; 300

Supplemental Fig. S8F). 301

When we analysed the inflorescences of 4xaly plants we observed that approximately 302

25% of the flowers showed an abnormal morphology including altered number of petals 303

(ranging from 3 to 7; Fig. 7A–C), markedly increased number of trichomes on the sepals and 304

severely reduced length of the stamen filaments (Fig. 7D–F). Detailed microscopy 305

examination of cleared 4xaly ovules demonstrated that a fraction of the ovules (28%; n = 317 306

ovules) showed diverse developmental defects, whereas others were indistinguishable from 307

Col-0 ovules (Fig. 7G-K). The 4xaly ovule phenotype was comprised of arrested female 308

gametophyte development or collapsed female gametophytes (16% 1.12 (mean SE)) and 309

abnormal integument development (12% 0.37 (mean SE)). Notably, the observed ovule 310

phenotypes did not correlate with the abnormal flower morphology. In contrast to the severe 311

developmental defects observed for a portion of the ovules, no altered phenotypes were 312

detected for pollen grains and germinated pollen tubes. Based on light microscopy analyses 313

and DAPI staining, the pollen grains released from 4xaly anthers had a wild-type appearance 314

(Supplemental Fig. S9). The aly1 T-DNA insertion line is in the quartet (qrt1-2) background, 315

therefore 4xaly anthers produce microspores that fail to separate during pollen development, 316

due to the lack of a pectin methylesterase in qrt1-2 (Francis et al. 2006). In vitro germination 317

of 4xaly pollen tetrads revealed that their germination is as efficient as Col-0 pollen, 318

indicating that they are fully viable (Supplemental Fig. S9). However, while performing the 319

pollen germination assays, we observed that the pollen is not as easily shed from dehiscent 320

4xaly anthers as pollen of dehiscent Col-0 anthers. Furthermore, the phenotype of 321

compromised pollen dispersal is independent from the severely reduced preanthesis filament 322

elongation observed in a portion of 4xaly flowers (Fig. 7F). 323

Compared with siliques of self-pollinated Col-0 flowers (Fig. 7L), 4xaly siliques were 324

shorter and the seed set was reduced, which was readily identified by gaps between developed 325

seeds (Fig. 7M). Counting the gaps in almost mature siliques derived from self-pollinated 326

flowers revealed 22.85% 3.63 (mean SE; n = 11) non-developed seeds in 4xaly siliques, 327

compared with 2.62% 0.97 (mean SE; n = 10) non-developed seeds in Col-0 siliques. 328

Subsequently, we performed hand-pollination experiments to determine whether the male or 329

the female or both contribute to the observed reproductive defects in 4xaly siliques. When we 330

hand-pollinated Col-0 pistils with 4xaly pollen, the seed set was comparable to that of Col-0 331

pistils that were hand-pollinated with Col-0 pollen (Fig. 7N, O). This suggests that the 332



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reduced stamen length of 4xaly anthers and/or the compromised pollen dispersal but not a 333

reduced fertility of 4xaly pollen contribute to the reproductive defects in 4xaly plants. 334

Furthermore, the hand-pollination of 4xaly pistils either with Col-0 or 4xaly pollen showed 335

that the reduced seed set in 4xaly siliques mainly originated from ovules with abortive female 336

gametophytes. Independent of the pollen donor plant, both the length of 4xaly siliques and the 337

seed set was comparable (Fig. 7P, Q). As expected, the reciprocal crossings of Col-0 pistils 338

with 4xaly pollen and 4xaly pistils with Col-0 pollen produced uniformly heterozygous 339

progeny (n = 15, each) for all four ALY genes. 340

341

The absence of conserved ALY factors in 4xaly plants affects nucleo-cytosolic mRNA 342

transport 343

To examine whether an Arabidopsis ALY protein can functionally replace the yeast 344

orthologue Yra1, a vector driving the expression of ALY1 under control of the GPD promoter 345

was introduced into a Yra1 shuffle strain (yra1::HIS3, pURA3-Yra1; Sträßer and Hurt 2000). 346

Colonies formed after several days when transformants were grown on 5-FOA-containing 347

plates (to select for colonies that had lost the pURA3-Yra1 plasmid), whereas the shuffle 348

strain transformed with the empty plasmid did not grow (Fig. 8A). Therefore, expression of 349

ALY1 can restore growth of the otherwise non-viable yra1 strain. However, as seen with the 350

similar complementation experiment performed using mouse ALY (Sträßer and Hurt 2000), 351

the complementation appears relatively inefficient (formation of small, slow growing 352

colonies) compared to the strain expressing Yra1 under control of the GPD promoter (Fig. 353

8A). 354

Finally, to analyse whether mRNA export is affected in 4xaly plants, in situ 355

hybridisation with a fluorescently labelled oligo(dT) probe was used to detect bulk poly(A) 356

mRNA. Using this method (Gong et al. 2005), the nucleo-cytosolic distribution of 357

polyadenylated mRNA was examined in seedling root cells. The fluorescent signals were 358

comparatively analysed by CLSM for Col-0, 4xaly and thp1 plants, since plants deficient in 359

the THP1 subunit of the TREX-2 complex show severe defects in mRNA export (Lu et al. 360

2010) and thus thp1 served as a reference for the experiment. When compared with Col-0, 361

stronger fluorescent signals were observed in the nuclei of thp1 cells (Fig.8A). With 4xaly 362

cells the nuclear fluorescence relative to the cytosolic signal was also clearly enhanced, albeit 363

not quite to the same level as for thp1 cells (Fig. 8B). These in situ hybridisation experiments 364

revealed an increased nuclear retention of polyadenylated mRNAs in 4xaly (and thp1) relative 365



13

to that in Col-0. Therefore, the ALY proteins (like THP1 (Lu et al. 2010)) play a role in the 366

export of mRNAs from plant nuclei. 367

368

DISCUSSION 369

The export of mRNAs from the nucleus to the cytosol for translation is a critical step in the 370

expression of protein-coding genes and this process is poorly characterised in plants. In yeast 371

and metazoa, mRNA export adaptors such as ALY proteins play a central role in nucleo-372

cytosolic mRNA transport. A characteristic feature of mRNA export adaptors is their RNA-373

binding activity (Walsh et al. 2010). In MST assays, Arabidopsis ALY1 bound in solution 374

with higher affinity to ssRNA when compared to its binding to dsRNA and ssDNA. The 375

interaction of mammalian ALY and yeast Yra1 with ssRNA was demonstrated using UV 376

crosslinking, electrophoretic mobility shift assays (EMSAs) and NMR spectroscopy (Sträßer 377

and Hurt 2000; Stutz et al. 2000; Zenklusen et al. 2001; Golovanov et al. 2006; Katahira et al. 378

2009). Consistent with our MST measurements, preferential binding to ssRNA was also seen 379

with murine ALY, as its binding was competed more efficiently by the addition of tRNAs 380

than by ssDNA (Stutz et al. 2000). In EMSAs, the variable N- and C-terminal domains of 381

mammalian ALY and yeast Yra1 were found to bind ssRNA, whereas no protein/RNA 382

interactions were detected for the individual RRM (Rodrigues et al. 2001; Zenklusen et al. 383

2001). Using NMR chemical shift mapping and UV cross-linking, it was observed that the 384

isolated RRM of mammalian ALY weakly bound to ssRNA, but high-affinity RNA 385

interaction required the variable N- and C-terminal domains (Golovanov et al. 2006). In 386

another EMSA study, the minimal RNA-binding site was mapped to a stretch of 21 amino 387

acid residues within the variable N-terminal domain of murine ALY (Hautbergue et al. 2008), 388

but this region is not well conserved in the Arabidopsis ALY sequences. Our MST analyses of 389

ALY1 RNA-binding revealed that the individual RRM interacts with ssRNA weakly and that 390

both the N- and C-terminal domains increase the affinity of the protein for RNA, whereby the 391

N-terminal domain has a greater effect. However, apparently RNA-binding sites scattered 392

over the length of ALY1 are required for its full capacity to interact with RNA. The presence 393

of separate RNA-binding sites would provide ALY proteins with the potential to interact with 394

different parts of an RNA molecule, which may play an important role in the packaging of 395

mRNPs (Heath et al. 2016). Like mammalian ALY (Yang et al. 2017), Arabidopsis ALY1 396

exhibits a significantly higher affinity for m5C-modified RNA. The m

5C modification is 397

suggested to play an essential role in mRNA export and other post-transcriptional processes in 398



14

mammals (Yang et al. 2017), a mechanism that may be conserved in plants (David et al. 399

2017). 400

Metazoan ALY and yeast Yra1 can be recruited to mRNAs co-transcriptionally by the 401

THO/TREX complex and the interaction is mediated by the DEAD-box RNA helicase 402

UAP56/Sub2. Additional ways of ALY loading onto mRNAs are through the 5′ cap binding 403

complex and the polyadenylation machinery (Wickramasinghe and Laskey 2015; Heath et al. 404

2016). ALY proteins were affinity-purified from Arabidopsis suspension culture cells together 405

with the THO core component TEX1 and with UAP56 (Sørensen et al. 2017), and ALY2 406

bound directly to UAP56 in vitro (Kammel et al. 2013). Here, ALY proteins interacted with 407

UAP56 in the yeast two-hybrid assay and FRET-APB experiments. Taken together, these 408

findings indicate that UAP56-mediated recruitment through the THO/TREX complex is 409

conserved for the four Arabidopsis ALY proteins. The Arabidopsis genome contains two 410

genes encoding identical UAP56 proteins, but likely no additional paralogue (Kammel et al. 411

2013). Hence, the THO/TREX interaction with ALY proteins in Arabidopsis might be 412

mediated exclusively by the RNA helicase UAP56. 413

The fluorescence microscopy analysis of plants expressing ALY-GFP fusion proteins 414

under control of their respective native promoters in an aly mutant background revealed that 415

the four ALY proteins are widely expressed throughout the plant. ALY1–4 were detected in 416

the nuclei of all root tip cells and leaf epidermis cells as well as stomatal guard cells. 417

Metazoan ALY and yeast Yra1 are also exclusively nuclear proteins, as they are displaced 418

from the mRNA before nucleo-cytosolic translocation (Walsh et al. 2010; Wickramasinghe 419

and Laskey 2015). Closer inspection of the subnuclear localisation of ALY proteins in leaf 420

cells, root cells and haploid cells of the female gametophyte revealed some differences. In 421

many root cells, ALY3/4 were detected weakly in the nucleoplasm with a strong enrichment 422

in nucleoli, whereas ALY1/2 were prominently present in the nucleoplasm and appear to be 423

excluded from nucleoli. Whereas ALY1/2 displayed a comparable subnuclear distribution in 424

root and leaf cells, the nucleolar enrichment of ALY3/4 was observed neither in leaf cells nor 425

in cells of the female gametophyte. Nucleolar localisation of the ALY mRNA export factors 426

may seem surprising, but, in addition to rRNA synthesis/processing, nucleoli play important 427

roles in the processing of other types of RNA including mRNA splicing and export (Shaw and 428

Brown 2012; Meier et al. 2017). It was demonstrated that in metazoa and particularly in the 429

best studied model yeast, RNAs other than mRNAs are exported by different mechanisms 430

involving exportins (Crm1 in yeast) (Köhler and Hurt 2007). rRNAs are exported from the 431



15

nucleus in form of large pre-ribosomes (pre-60S, pre-40S), which in yeast is essentially 432

mediated by Crm1 and the adaptor protein Nmd3 (Peña et al. 2017). Both exportins and 433

Nmr3-like proteins are conserved in plants, and Arabidopsis NMD3 is required for export of 434

ribosomal subunits (Merkle 2011; Chen et al. 2012). Therefore, it is unlikely that plant ALY 435

proteins act in the export of pre-ribosomes. Previous analyses addressing the subcellular 436

localisation of Arabidopsis ALY-GFP fusion proteins utilised transient fusion protein 437

expression driven by viral promoters either in N. benthamiana leaves (Uhrig et al. 2004) or in 438

Arabidopsis suspension culture cells (Pendle et al. 2005), yielding somewhat different results 439

especially for ALY1, ALY3 and ALY4. The most striking differences include a remarkable 440

enrichment of ALY1 in nucleoli, a very prominent ring-shaped staining of the nucleolar 441

periphery particularly with ALY3/4, and generally that the ALY proteins are often localised 442

to speckles and foci within the nucleoplasm. The comparable subnuclear distribution of ALY1 443

and ALY2 versus ALY3 and ALY4 observed in root and leaf cell nuclei of stably transformed 444

Arabidopsis plants is in line with the degree of amino acid sequence similarity of ALY1 and 445

ALY2 versus ALY3 and ALY4 (Supplemental Fig. S1 and S2), which is also reflected by the 446

conservation of their respective gene structures (Supplemental Fig. S3). 447

Whereas the ALY-GFP fusion proteins appeared to be ubiquitously expressed in roots 448

and leaves, the analysis of reproductive tissues revealed interesting differences regarding their 449

expression pattern and expression level. In pollen grains, the expression of ALY3 is weaker 450

and differs from that of ALY1/2, since it was only detectable in the nucleus of the pollen 451

vegetative cell, whereas ALY4 was not expressed at all. However, despite expression of 452

ALY1–3 in the pollen vegetative cell, neither pollen development nor pollen germination, or 453

pollen tube growth, was noticeably affected when 4xaly pollen were grown in vitro, or when 454

they were used for hand-pollination experiments. These observations suggest that ALY-455

mediated mRNA export either does not play a critical role in pollen, or that mRNA export 456

from the nucleus of the pollen vegetative cell is primarily achieved by additional, yet 457

uncharacterised mRNA export factors (see below). Furthermore, our hand pollination 458

experiments using Col-0 pistils also demonstrate that 4xaly sperm cells are fully functional. 459

Although ALY1 and ALY2 are expressed in sperm cells, seed development of ovules fertilised 460

with 4xaly sperm cells was not reduced. Thus, male reproductive 4xaly phenotypes are only 461

caused by a significantly reduced length of stamen filaments and the compromised release of 462

pollen from dehiscent anthers. In the ovule, ALY1 was restricted to the female gametophyte, 463

whereas the other ALY proteins were also expressed in the surrounding sporophytic tissues. 464



16

Around 16% of 4xaly ovules showed developmental defects in the female gametophyte, 465

whereas the development of the integuments was affected in 12% of the mutant ovules. 466

Considering these phenotypes, the expression pattern of ALY proteins suggests that all four 467

ALY proteins act redundantly during female gametogenesis. On the other hand, the joint 468

expression of ALY2, ALY3 and ALY4 in the sporophytic tissues of the ovule implies their 469

contribution to the anatomically correct development of the integuments. Nevertheless, the 470

phenotypic changes in 4xaly ovules occurred with low penetrance, suggesting that ALY 471

proteins act redundantly with additional mRNA export factors during female gametophyte 472

development. Since we also found filament elongation and pollen dispersal to be 473

compromised in 4xaly stamens, cross-pollination experiments were necessary to clarify the 474

reason(s) for the reduced seed set of self-pollinated 4xaly pistils. These experiments clearly 475

showed that the ovules with abortive female gametophytes provoke the reduced seed set in 476

4xaly siliques, whereas 4xaly pollen is fully fertile when all physical restraints are overcome. 477

In metazoa, the number of genes encoding different ALY proteins ranges from one in 478

humans and two in D. melanogaster to three in C. elegans (Gatfield and Izaurralde 2002; 479

Longman et al. 2003; Katahira et al. 2009). The ALY genes of flowering plants seem to be 480

more diversified, as the analysed monocot and dicot plant genomes encode four or five 481

different ALY proteins. Inactivation of the human ALY in HeLa cells, and similarly the 482

depletion of ALY in D. melanogaster SL2 cells, resulted in a mild defect in the export of bulk 483

mRNAs (Gatfield and Izaurralde 2002; Katahira et al. 2009), whereas the simultaneous down-484

regulation of the three ALY proteins in C. elegans did not cause detectable nuclear 485

accumulation of polyadenylated mRNAs (Longman et al. 2003). In agreement with these 486

findings, in Arabidopsis 4xaly plants, nuclear accumulation of polyadenylated mRNAs was 487

observed, but not a complete block of mRNA export. Therefore, as in metazoa, there is some 488

redundancy among mRNA export adaptors (Wickramasinghe and Laskey 2015; Heath et al. 489

2016) and, in addition to ALYs, other proteins may serve this function in Arabidopsis. 490

Possible candidates for additional mRNA export adaptors include certain SR proteins and/or 491

UIF-like proteins (Heath et al. 2016). D. melanogaster cells lacking ALY proteins show a 492

mild proliferation defect, and simultaneous depletion of the three ALYs in C. elegans resulted 493

only in a slight decrease of larval mobility (Gatfield and Izaurralde 2002; Longman et al. 494

2003). Compared to these findings, 4xaly plants exhibited various defects in vegetative and 495

reproductive development, whereas aly single mutants as well as aly1 aly2 and aly3 aly4 496

double mutants displayed essentially a wild-type appearance. These results indicate that there 497



17

is functional redundancy between the different ALY proteins and that the phenotypic defects 498

of 4xaly plants may be caused by cumulative effects on mRNA export that are not detectable 499

in the single and double mutants. However, the fact that flowering plants generally express 500

several ALY proteins argues against simple redundancy, and suggests that there is (partial) 501

functional specialisation. This is supported by the observations that Arabidopsis ALY proteins 502

are differently expressed, at least in reproductive tissue, and that ALY1/2 display subnuclear 503

localisation that is distinct from that of ALY3/4 in root and leaf cells. Moreover, there is the 504

possibility that various ALY proteins are involved in the nucleo-cytosolic transport of 505

different subsets of mRNAs. Since this could be combined with tissue- and/or developmental 506

stage-specific effects, analysing these options will be a challenge for future investigations. 507

508

CONCLUSION 509

In plants, only few components of the mRNA export machinery have been functionally 510

characterised to date. These include some nucleoporins of the nuclear pore complex (Parry 511

2015; Meier et al. 2017) and the TREX-2 complex that localises to the nucleoplasmic side of 512

nuclear pores (Lu et al. 2010). Moreover, subunits of the THO/TREX core complex as well as 513

the associated MOS11 protein are involved in nucleo-cytosolic mRNA transport in 514

Arabidopsis (Germain et al. 2010; Pan et al. 2012; Xu et al. 2015; Sørensen et al. 2017). The 515

data presented here demonstrate that the ALY proteins of flowering plants are more 516

diversified than those in other organisms and that they are part of the plant mRNA export 517

pathway, likely acting as mRNA export adaptors. This notion is supported by their nuclear 518

localisation, their RNA-binding activity, their UAP56-mediated interaction with the 519

THO/TREX complex, that expression of ALY1 can complement growth of the non-viable 520

yeast yra1 mutant and, most importantly, by the nuclear mRNA accumulation in the absence 521

of the four ALY proteins. However, it remains to be seen whether ALY proteins functionally 522

interact with the thus far unidentified plant mRNA export receptor. Finally, the phenotype of 523

plants lacking ALY proteins illustrates that impaired mRNA export can cause severe defects 524

in vegetative and reproductive development. 525

526

527

METHODS 528

Plant material and documentation 529



18

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). 532

After sowing, seeds were stratified in darkness for 48h at 4°C prior to incubation in the plant 533

growth chamber. Age of plants is stated as days after stratification (DAS). For root analyses 534

plants were grown on vertically oriented MS plates in a plant incubator (Percival Scientific) 535

under long-day conditions as described before (Dürr et al. 2014). Seeds of the aly T-DNA 536

insertion lines were obtained from the European Arabidopsis stock centre 537

(http://www.arabidopsis.info/) and those of thp1-3 (Lu et al. 2010) were a gift from Yuhai 538

Cui. Mutant plants were characterised by PCR-based genotyping in combination with 539

sequencing of the insertion region and by RT-PCR (Antosz et al. 2017). By crossing the 540

parental lines as previously described (Lolas et al. 2010) higher-order mutant lines were 541

generated. Using plasmids harbouring different ALY-GFP (or UAP56-GFP) fusion constructs 542

under control of the respective native promoters (Supplementary Table S2) and 543

Agrobacterium-mediated floral dip transformation as previously described (Pedersen et al. 544

2010; Antosch et al. 2015), plants were generated that express ALY-GFP (or UAP56-GFP). 545

Plant phenotypes were documented and quantified as previously described (Dürr et al. 2014; 546

Antosz et al. 2017). All phenotypic analyses were independently performed at least twice and 547

representative examples of the reproduced experiments are shown. 548

549

Preparation of pollen grains and pollen germination 550

Mature pollen grains were mounted in DAPI solution (6 µg/ml 4′,6-diamidin-2-phenylindol in 551

0.1 M PBS, pH 7.4) and imaged by fluorescence microscopy. Pollen grains were germinated 552

as previously described (Vogler et al. 2014). 553

554

Preparation of ovules and siliques 555

Ovules were dissected and mounted as previously described (Sprunck et al. 2012) using 556

unpollinated pistils at floral stage 12 (Smyth et al. 1990). Ovules were dissected on glass 557

slides, either in chloral hydrate/glycerol/water (8:1:2, w/v/v) for differential interference 558

contrast (DIC) microscopy, or in 50 mM sodium phosphate buffer (pH 7.5) for confocal laser 559

scanning microscopy (CLSM). Emasculated pistils at floral stage 12 were used for cross-560

pollination experiments. Seed set of self-pollinated and 11-day-old cross-pollinated siliques 561

was investigated as described (Sprunck et al. 2012). 562


http://www.arabidopsis.info/


19

563

Plasmid constructions 564

The required gene or cDNA sequences were amplified by PCR with KAPA DNA polymerase 565

(PeqLab) using Arabidopsis genomic DNA or cDNA as template and the primers (providing 566

also the required restriction enzyme cleavage sites) listed in Supplemental Table S2. The PCR 567

fragments were inserted into suitable plasmids using standard methods. All plasmid 568

constructions were checked by DNA sequencing, and details of the plasmids generated in this 569

work are summarised in Supplemental Table S2. 570

571

Production of recombinant proteins 572

For MST experiments, full-length ALY1 and truncated versions of the protein (Supplemental 573

Table S2) were expressed in E. coli using plasmid pET24b-GB1 (Hautbergue et al. 2008) for 574

the expression of the ALY1 proteins fused to a 6xHis-tag for metal-chelate purification and a 575

GB1-tag increasing protein stability/solubility (Gronenborn et al. 1991). The proteins were 576

isolated from E. coli lysates by metal-chelate affinity chromatography using Ni-NTA beads 577

(Qiagen) as previously described (Kammel et al. 2013). Eluted ALY1 proteins were further 578

purified by FPLC ion exchange chromatography using a Resource Q column (GE Healthcare) 579

equilibrated in buffer D (10 mM sodium phosphate, pH 9.0, 1 mM EDTA, 1 mM DTT, 0.5 580

mM PMSF) (Grasser et al. 1996). Proteins were eluted with a linear gradient of 0–1 M NaCl 581

in buffer D and ALY1 containing fractions were pooled and passed through PD-10 columns 582

(GE Healthcare) changing to protein buffer (50 mM Tris/HCl, pH 8.0, 100 mM NaCl, 10 mM 583

2-mercaptoethanol, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, 0.5 mM PMSF, 50 mM L-584

Arg, 50 mM L-Glu (Golovanov et al. 2004)). Finally, proteins were characterised by SDS-585

PAGE in combination with Coomassie staining and by mass spectrometry. 586

587

Yeast two-hybrid and complementation assays 588

Yeast two-hybrid assays were essentially performed according to the manufacturer’s 589

instructions (Clontech). In brief, yeast cells of the strain AH109 were co-transformed with 590

pGBKT7 and pGADT7 plasmids (Supplemental Table S2) and grown at 30°C on SD/-Leu/-591

Trp (double drop-out, DDO) medium. For interaction assays, cells were grown on SD/-Ade/-592

Leu/-Trp/-His (quadruple drop-out, QDO) medium for 2 days (Kammel et al. 2013). As 593

positive and negative controls served the interactions between p53 and the SV40 large T-594

antigen, and between lamin and the SV40 large T-antigen, respectively, provided by the 595



20

manufacturer. Using the YRA1 shuffle strain (yra1::HIS3, pURA3-Yra1 (Sträßer and Hurt 596

2000)) it was tested whether ALY1 expressed under control of the GPD promoter using the 2µ 597

p425-GPD vector (Mumberg et al. 1995) can replace Yra1, which was performed as 598

previously described (Sträßer and Hurt 2000) by ultimately growing the complemented cells 599

on 5´-fluoroorotic acid (5-FOA) plates. 600

601

PCR-based genotyping and reverse-transcription PCR (RT-PCR) 602

To distinguish between plants being wild type, heterozygous, or homozygous for the T-DNA 603

insertions, genomic DNA was isolated from leaves. The genomic DNA was used for PCR 604

analysis with Taq DNA polymerase (PeqLab) and primers specific for T-DNA insertions and 605

the target genes (Supplemental Table S2). For RT-PCR, total RNA was extracted from ~100 606

mg of frozen plant tissue using the TRIzol method (Invitrogen), before the RNA samples were 607

treated with DNAse. Reverse transcription was performed using 2 μg of RNA and Revert Aid 608

H minus M-MuLV reverse transcriptase (Thermo Scientific). The obtained cDNA was 609

amplified by PCR using Taq DNA polymerase (PeqLab) as previously described (Dürr et al. 610

2014). 611

612

Fluorescent MicroScale Thermophoresis (MST) binding assay 613

MicroScale Thermophoresis (MST) binding experiments were carried out essentially as 614

previously described (Kammel et al. 2013) with 200 nM 25-nt Cy3-labeled ssRNA, dsRNA or 615

ssDNA oligonucleotides or 19-nt Cy3-labeled ssRNA with or without m5C-modification 616

(Yang et al. 2017) (Eurofins Genomics, Supplemental Table S2). MST measurements were 617

performed in protein buffer with a range of concentrations of ALY1 proteins at 40% MST 618

power, 50% LED power in standard capillaries at 25°C on a Monolith NT.115 device 619

(NanoTemper Technologies). The fluorescence was monitored and used for data analysis in 620

Thermophoresis and TJump. To calculate the fraction bound, the ΔFnorm value of each point is 621

divided by the amplitude of the fitted curve, resulting in values from 0 to 1 (0 = unbound, 1 = 622

bound), and processed using the KaleidaGraph 4.5 software and fitted using the KD fit 623

formula derived from the law of mass action. Differences between data sets were statistically 624

evaluated using an unpaired Student’s t-test. 625

626

Light microscopy 627



21

For analysis of GFP fusion protein expression, plants of at least three independent transgenic 628

lines each harbouring the different ALY-GFP fusion constructs in the respective mutant 629

background were selected for further analysis based on RT-PCR analysis indicating uniform 630

expression levels of ALY similar to Col-0 (Antosch et al. 2015). Imaging of GFP fluorescence 631

was performed on an inverted Leica SP8 CLSM with a 40x/1.3 NA and a 60x/1.3 NA oil 632

immersion objective. GFP was excited using the 488 nm argon laser line, emission was 633

detected from 495 to 545 nm (roots, leaves) or 500 to 535 nm (ovules, pollen) by a hybrid 634

detector. Propidium iodide staining was performed as previously described (Dürr et al. 2014). 635

Flowers and cleared siliques were imaged using a stereomicroscope SteREO Discovery.V8 636

(Zeiss). Cleared ovules and DAPI-stained pollen grains were investigated at the APOTOME 637

FL (Zeiss) with a DIC objective 40x/1.4 oil and a 49DAPI reflector for DAPI fluorescence. 638

Germinated pollen tubes were analysed using an inverse NIKON Eclipse 2000-S microscope 639

equipped with a ZEISS Axio-Cam MRm CCD camera using a 209/0.4 NA air objective. 640

641

Förster resonance energy transfer (FRET) 642

FRET acceptor photobleaching (FRET-APB) was essentially performed as previously 643

described (Weidtkamp-Peters and Stahl 2017) using a SP8 microscope (Leica). Briefly, a 644

square (0.5 x 0.5 cm) of an infiltrated Nicotiana benthamiana leaf was mounted in H2O on an 645

objective slide with the abaxial side facing up. A circular area of 12 μm was bleached at 100% 646

laser power, for 80 iterations. 15 pre-bleach and 15 post-bleach images were analysed by 647

ImageJ. The mean FRET efficiency was calculated as follows: ((IPOST - IPRE) / IPRE) x 648

100. With IPRE / IPRE = mean fluorescence intensity of 15 pre-bleached / post-bleached 649

frames. 650

651

Whole mount in situ hybridisation 652

To determine the relative distribution of bulk mRNA in nuclei and cytosol a previously 653

described protocol was adopted (Gong et al. 2005) using 6-day-old seedlings grown on solid 654

MS medium. Hybridisation was performed in PerfectHyb plus solution (Sigma) with an Alexa 655

Fluor 488-labelled 48-nt oligo(dT) probe. Fluorescent signals of seedling roots were analysed 656

using CLSM with a Leica SP8 microscope (Sørensen et al. 2017). 657

658

Accession Numbers 659



22

Sequence data for the genes described in this article can be found in the Arabidopsis Genome 660

Initiative or GenBank/EMBL databases under the following accession numbers: ALY1 661

(At5g59950), ALY2 (At5g02530), ALY3 (At1g66260), ALY4 (At5g37720), UAP56A 662

(At5g11170), UAP56B (At5g11200). 663

664

Supplemental Data 665

The following Supplemental materials are available. 666

Supplemental Figure S1. Amino acid sequence identity and alignment of Arabidopsis ALY 667

proteins. 668

Supplemental Figure S2. Sequence similarity of ALY sequences 669

Supplemental Figure S3. Molecular characterisation of aly T-DNA insertion mutants. 670

Supplemental Figure S4. ALY transcript levels in aly mutant plants expressing ALY-GFP 671

fusion proteins. 672

Supplemental Figure S5. Subnuclear distribution of mutant ALY1 and ALY3. 673

Supplemental Figure S6. ALY-GFP signals in growing pollen tubes, analysed by CLSM. 674

Supplemental Figure S7. Phenotype of aly single- and double-mutant plants. 675

Supplemental Figure S8. Phenotypic characterisation of 4xaly plants relative to Col-0. 676

Supplemental Figure S9. The pollen grain phenotype and pollen germination efficiency of 677

the 4xaly mutant is similar to that of the wild type. 678

Supplemental Table S1. Relative subnuclear localisation of the different ALY-GFP proteins 679

in root cells. 680

Supplemental Table S2. Oligonucleotide primers used in this study and construction of 681

plasmids. 682

683

FIGURE LEGENDS 684

Figure 1. Production of recombinant ALY1 proteins and MST analysis of their interaction 685

with nucleic acids. A, Schematic representation of ALY1 depicting the different domains of 686

the protein. The RNA recognition motif (RRM) and the relatively variable N- and C-terminal 687

domains (cf. Supplemental Fig. S1) are indicated. Numbers indicate amino acid sequence 688

positions that delimit the domains. B, SDS-PAGE analysis of purified full-length and 689

truncated ALY1 expressed in E. coli, alongside the unfused 6xHis-GB1 tag. C, MST analysis 690

of the interaction of full-length ALY1 with different nucleic acids. Incubation of the unfused 691

6xHis-GB1-tag with ssRNA was included as a control. D, MST analysis of the interaction of 692



23

full-length and truncated versions of ALY1 with ssRNA. E, MST analysis of the interaction 693

of full-length ALY1 with m5C-modified 19-nt ssRNA and with the unmodified control RNA. 694

Each data point represents two biological and two technical replicates, and error bars indicate 695

standard deviation of the biological replicates. 696

697

Figure 2. ALY proteins interact with UAP56 in yeast two-hybrid assays and FRET analyses. 698

A, Yeast two-hybrid assays with cells harbouring the indicated constructs grown on double 699

drop-out (DDO, left panels) or quadruple drop-out (QDO, right panels) media. B, Protein 700

interactions analysed by FRET-APB. N. benthaniana leaves were co-infiltrated with vectors 701

expressing donor (eGFP, ALY1, ALY3; green)/acceptor (mCherry, UAP56; red) 702

combinations as indicated, alongside positive and negative controls. Transiently transformed 703

cells were analysed using two biological replicates by acceptor-photobleaching FRET. Mean 704

FRET-APB efficiencies (SD, 8 analysed nuclei each) are shown. *** indicates statistically 705

significant difference (Student´s t-test, P < 0.001). 706

707

Figure 3. Localisation of ALY1–4- and UAP56-GFP fusion proteins in nuclei of root and leaf 708

cells. A, Root tips of 8 DAS plants expressing the indicated GFP fusion proteins under control 709

of the respective native promotors. GFP fluorescence is visible in green, while propidium 710

iodide staining is in red. B, Root tips as in A shown at higher magnification. C, Abaxial 711

surfaces of second leaf pair from 14 DAS plants depicting GFP fluorescence in nuclei of 712

epidermal cells and of stomatal guard cells. D, Magnified images of guard cells with nuclear 713

GFP fluorescence. Bars = 50 µm (A, B), 25 µm (C), or 10 µm (D). 714

715

Figure 4. Subnuclear localisation of ALY1–4- and UAP56-GFP fusion proteins in nuclei of 716

root and leaf cells. A, Root cell nuclei from 8 DAS plants expressing the indicated GFP fusion 717

proteins under control of the respective ALY native promotors. The images depicted represent 718

the most commonly observed subnuclear distributions of fusion protein. B, Cell nuclei from 719

the second leaf pair of 14 DAS plants of the same GFP fusion protein lines depicted in A. n = 720

nucleoli. Bars = 5 μm. 721

722

Figure 5. Localisation of ALY-GFP fusion proteins in pollen grains and ovules. A, mature 723

pollen grains. Filled and open arrowheads indicate sperm cell nuclei and vegetative nuclei, 724

respectively. DNA is counterstained with DAPI (blue). GFP fluorescence (left panels) and the 725



24

corresponding bright field image, merged with fluorescent signals (right panels), is shown. B, 726

Mature unfertilised ovules. GFP fluorescence (left panels) and the corresponding bright field 727

image, merged with GFP signals (right panels), is shown. Insets depict ALY-GFP signals in 728

the nuclei of female gametophytic cells. Filled and open arrowheads and asterisks indicate 729

synergid nuclei, egg cell nuclei, and central cell nuclei, respectively. Bars = 5 µm (A) or 25 730

µm (B). 731

732

Figure 6. Phenotype of the quadruple aly mutant plants (4xaly) relative to Col-0. 733

Representative images of plants at 28 DAS (A) and 42 DAS. (B) plants grown under long-day 734

conditions in soil. (C) Roots of 8 DAS plants grown on solid MS medium. 735

736

Figure 7. 4xaly plants produce a number of flowers with altered morphology and abnormal 737

ovules. A–F, Flower morphology. A, Col-0 flower depicting normal petal number. B, A 4xaly 738

flower depicting altered petal number. C, Normal 4xaly flower as observed in the majority of 739

cases. D, Col-0 flower depicting normal trichome number on sepals and stamen filament 740

length (filled arrowheads). E and F, Abnormal 4xaly flower depicting increased trichome 741

number on sepals (open arrowheads) and reduced stamen filament length (filled arrowheads). 742

G–K, Differential contrast microscopy of cleared ovules. G, Col-0 ovule depicting normal 743

morphology. H, 4xaly ovule depicting normal morphology. I, 4xaly ovule depicting arrested 744

female gametophyte development. J, 4xaly ovule depicting collapsed female gametophyte. K, 745

4xaly ovule depicting abnormal integument development. Filled and open arrowheads and 746

asterisks indicate synergid cell nuclei, egg cell nuclei, and central cell nuclei, respectively. FG 747

= female gametophyte. II = inner integuments. OI = outer integuments. L and M, Morphology 748

of self-pollinated siliques of the indicated genotypes (pistil x pollen). N–Q, Morphology of 749

cross-pollinated siliques of the indicated genotypes (pistil x pollen). Open arrowheads in M 750

indicate gaps with undeveloped seeds in a 4xaly silique. Bars = 0.2 mm (A–F), 20 µm (G–K), 751

or 2 mm (L–Q). 752

753

Figure 8. Complementation of the yeast yra1 mutant and analysis of nucleo-cytosolic 754

distribution of polyadenylated mRNA in 4xaly plants. A, Growth of the YRA1 shuffle strain 755

(yra1::HIS3, pURA3-Yra1) following complementation with Arabidopsis ALY1 or yeast 756

YRA1, alongside an empty vector negative control. For ALY1, three individual transformants 757

were analysed. Cells were grown on 5-FOA plates, thus allowing only growth of yra1::HIS3 758



25

cells that have lost the pURA3-Yra1 plasmid. The plate was photographed after 18 days at 759

24ºC to better visualise small, slow growing colonies. B, Root cells of 6 DAS Col-0, 4xaly 760

and thp1 seedlings examined using whole mount in situ hybridisation with a fluorescently 761

labelled 48-nt oligo (dT) probe. Confocal laser scanning microscopy images (taken using 762

identical microscope settings) of representative regions of the analysed roots (top panels) and 763

individual cells (bottom panels) are shown. Bars = 60 μm (top panels) or 10 μm (bottom 764

panels). C, The fluorescent hybridisation signal of nuclei relative to cytosol as quantified for 765

58 nuclei from three roots of each genotype. The ratios are depicted relative to Col-0 (ratio: 766

1) with error bars indicating standard deviation. *** indicates statistically significant 767

difference (two-tailed Student´s t-test, P < 0.001). 768

769

770

771

ACKNOWLEDGEMENTS 772

We thank Anna Geiger, Valentin Bergér and Dominik Fiegle for contributions during early 773

stages of the project, Benedikt Müller for help with CLSM imaging, Astrid Bruckmann and 774

Eduard Hochmuth for mass spectrometry analyses, Antje Böttinger for technical assistance, 775

Stuart Wilson for providing plasmid pET24b-GB1, Ed Hurt for the yeast YRA1 shuffle strain 776

(yra1::HIS3, pURA3-YRA1), Yuhai Cui for the thp1-3 mutant line, the Nottingham 777

Arabidopsis Stock Centre (NASC) for providing Arabidopsis T-DNA insertion lines. 778

779

780

REFERENCES 781

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Antosz W, Pfab A, Ehrnsberger HF, Holzinger P, Köllen K, Mortensen SA, Bruckmann A, 785 Schubert T, Längst G, Griesenbeck J, Schubert V, Grasser M, Grasser KD. (2017) 786 Composition of the Arabidopsis RNA polymerase II transcript elongation complex reveals the 787 interplay between elongation and mRNA processing factors. Plant Cell 29:854-870 788

Bruhn L, Munnerlyn A, Grosschedl R. (1997) ALY, a context-dependent coactivator of LEF-1 and 789 AML-1, is required for TCRalpha enhancer function. Genes Dev 11:640-653 790

Burd CG, Dreyfuss G. (1994) Conserved structures and diversity of functions of RNA-binding 791 proteins. Science 265:615-621 792

Canto T, Uhrig JF, Swanson M, Wright KM, MacFarlane SA. (2006) Translocation of tomato 793 bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing 794 suppressor activity. J Virol 80:9064-9072 795



26

Chen MQ, Zhang AH, Zhang Q, Zhang BC, Nan J, Li X, Liu N, Qu H, Lu CM, Sudmorgen, 796 Zhou YH, Xu ZH, Bai SN. (2012) Arabidopsis NMD3 is required for nuclear export of 60S 797 ribosomal subunits and affects secondary cell wall thickening. PLoS One 7:e35904 798

David R, Burgess A, Parker B, Li J, Pulsford K, Sibbritt T, Preiss T, Searle IR. (2017) 799 Transcriptome-wide mapping of RNA 5-methylcytosine in Arabidopsis mRNAs and noncoding 800 RNAs. Plant Cell 29:445-460 801

Dufu K, Livingstone MJ, Seebacher J, Gygi SP, Wilson SA, Reed R. (2010) ATP is required for 802 interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to 803 assemble the TREX complex. Genes Dev 24:2043-2053 804

Dürr J, Lolas IB, Sørensen BB, Schubert V, Houben A, Melzer M, Deutzmann R, Grasser M, 805 Grasser KD. (2014) The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene 806 expression in Arabidopsis. Nucleic Acids Res 42:4332-4347 807

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933 934 935

936



Figure 1. Production of recombinant ALY1 proteins and MST analysis of their interaction with nucleic

acids. A, schematic representation of ALY1 depicting the different domains of the protein. Indicated are the

RRM and the relatively variable N- and C-terminal domains (cf. Supplemental Fig. S1). Numbers indicate

amino acid sequence positions delineating the domains. B, full-length ALY1 and truncated versions of the

protein (and the unfused 6xHis-GB1 tag) were expressed in E. coli, purified, and the SDS-PAGE analysis

of the purified proteins after Coomassie staining is shown. C, MST analysis of the interaction of full-length

ALY1 with different nucleic acids, as well as incubation of the unfused 6xHis-GB1-tag with ssRNA,

revealing no interaction. In these assays, ALY1 bound ssRNA with higher affinity than dsRNA and ssDNA

(P < 0.05 and P < 0.001, respectively). D, MST analysis of the interaction of full-length and truncated

versions of ALY1 with ssRNA, revealing that ALY1 displayed higher affinity for ssRNA than the truncated

versions of the protein (P < 0.005). E, MST analysis of the interaction of full-length ALY1 with m5C-

modified 19-nt ssRNA and with the unmodified control RNA, demonstrating that the m5C-modified RNA is

the preferred target (P < 0.01). Measurements were performed with each two biological and two technical

replicates, and error bars indicate standard deviation of the biological replicates.

A

RRM N-ter C-ter ALY1

1 85 164 245

–

– 15

–

– 30

50

70

B

C

fra

cti

on

bo

un

d

fra

cti

on

bo

un

d

fra

cti

on

bo

un

d

D E ALY1

ALY1RRM

ALY1ΔN

ALY1ΔC

ssRNA ssRNA-m5C



UAP56-ALY2

UAP56-BD

AD-ALY2

neg. control

pos. control

UAP56-ALY1

UAP56-BD

AD-ALY1

neg. control

pos. control

UAP56-ALY3

UAP56-BD

AD-ALY3

neg. control

pos. control

UAP56-ALY4

UAP56-BD

AD-ALY4

neg. control

pos. control

DDO QDO A

B



Figure 3. ALY1-4 and UAP56 GFP-fusion proteins are visualised by CLSM in nuclei of root and leaf cells.

A, root tips of 8 DAS plants expressing the indicated GFP fusion proteins under control of the respective

native promotors. GFP fluorescence is visible in green, while propidium iodide staining is in red. B, root

tips of the same plant lines shown at higher magnification. C, abaxial surfaces of second leaf pair from 14

DAS plants. GFP fluorescence is visible in nuclei of epidermal cells and of stomatal guard cells. D,

magnified images of guard cells with nuclear GFP fluorescence. Size bars represent 50 µm (A,B), 25 µm

(C), 10 µm (D).

ALY1-GFP ALY2-GFP ALY3-GFP ALY4-GFP UAP-GFP A

B

C

D



Figure 4. Subnuclear localisation of ALY1-4 and UAP56 GFP-fusion proteins visualised by CLSM in

nuclei of root and leaf cells. A, nuclei of root cells of 8 DAS plants expressing the indicated GFP

fusion proteins under control of the respective native promotors. Generally, the subnuclear

distribution of the ALY proteins exhibits some variability and the shown images represent the most

common observations. B, nuclei of cells of the second leaf pair of 14 DAS plants of the same plant

lines. The position of nucleoli is indicated by n and size bars represent 5 μm.

B NLS-GFP UAP56-GFP ALY1-GFP ALY2-GFP ALY3-GFP ALY4-GFP

A NLS-GFP UAP56-GFP ALY1-GFP ALY2-GFP ALY3-GFP ALY4-GFP

n n n

n n

n n n



*

ALY3-GFP

*

ALY4-GFP

*

ALY1-GFP

B

Figure 5. Expression of ALY-GFP fusion proteins in pollen grains and ovules, analysed by CLSM. A,

mature pollen grains. Filled arrowheads point at sperm cell nuclei, the vegetative nuclei are labelled by

open arrowheads. DNA is counterstained with DAPI (blue). GFP fluorescence (left panels) and the

corresponding bright field image, merged with fluorescent signals (right panels), is shown. B, mature

unfertilised ovules. GFP fluorescence (left panels) and the corresponding bright field image, merged with

GFP signals (right panels), is shown. Insets depict ALY-GFP signals in the nuclei of female

gametophytic cells. Filled arrowheads point at synergid nuclei; open arrowheads label egg cell nuclei;

asterisks label central cell nuclei. Scale bars represent 5 µm (A) or 25 µm (B).

ALY2-GFP

ALY3-GFP

ALY4-GFP

ALY1-GFP

A

ALY2-GFP

*



Col-0

4xaly

28 DAS A

Col-0 4xaly

42 DAS B

Col-0 4xaly

C 8 DAS

Figure 6. Phenotype of the quadruple aly mutant plants (4xaly) relative to Col-0. Representative

images of plants at 28 DAS (A) and 42 DAS (B) grown under long day conditions in soil. (C) Roots

of 8 DAS plants grown on solid MS medium.

aly4x



Figure 7. 4xaly plants partly produce flowers with altered morphology and abnormal ovules. Compared to Col-0

flowers (A), part of the 4xaly flowers have an altered number of petals (B), but the majority of the flowers exhibit

normal morphology (C). Another aspect of the altered flower morphology includes an increased number of

trichomes on the sepals (open arrowheads in E, F) and stamen with strongly reduced lengths of filaments (white

arrowheads in F) compared with the wild type (D). (G-K) Differential contrast microscopy of cleared ovules.

Compared to the wild type (G) 4xaly ovules showed either no phenotypic alterations (H), or diverse phenotypes

such as ovules with arrested female gametophyte development (I), collapsed female gametophytes (J), and

abnormal integument development (K). Black arrowheads point at synergid cell nuclei; open arrow heads label egg

cell nuclei; asterisks label central cell nuclei. Abbreviations: FG, female gametophyte, II, inner integuments, OI,

outer integuments. Self-pollinated (L, M) and cross-pollinated siliques (N-Q). Arrowheads in (M) point at gaps with

undeveloped seeds in a 4xaly silique. Genotypes of the crossings (pistil x pollen) are indicated. Scale bars

represent 0.2 mm (A-F), 20 µm (G-K) and 2 mm (L-Q).

WT Col-0 4xaly

A C

Col-0

D

4xaly

E

4xaly

F

*

4xaly

H FG

4xaly

I

OI OI

II

II

FG

4xaly

K

*

Col-0

G

4xaly

J FG

4xaly

B

P

4xaly x 4xaly

Q

4xaly x Col-0

L

Col-0

O

Col-0 x 4xaly

N

Col-0 x Col-0 4xaly

M



Figure 8. Complementation of the yeast yra1 mutant and analysis of nucleo-cytosolic distribution of

polyadenylated mRNA in 4xaly plants. A, restoration of growth of the yra1::HIS3 strain by expression of

Arabidopsis ALY1. The YRA1 shuffle strain (yra1::HIS3, pURA3-Yra1) was transformed with the 2μ plasmids

p425-GPD-ALY1 (+ALY1), p425-GPD-Yra1 (+Yra1) or empty p425-GPD (empty vector). In the case of ALY1,

three individual transformands were analysed. Finally, cells were grown on 5-FOA plates, on which only cells

can grow that have lost the pURA3-Yra1 plasmid, and thus are yra1::HIS3. The plate was photographed after

18 d at 24ºC. B, root cells of 6 DAS Col-0, 4xaly and thp1 seedlings were examined using whole mount in situ

hybridisation with a fluorescently labelled 48-nt oligo (dT) probe. CLSM images (taken using identical

microscope settings) of representative regions of the analysed roots (top panels) and individual cells (bottom

panels) are shown. Size bars represent 60 μm (top panels) and 10 μm (bottom panels). C, the fluorescent

hybridisation signal of nuclei relative to cytosol was quantified for 58 nuclei from three roots of each

genotype. The ratios are depicted relative to Col-0 (ratio: 1) with error bars indicating standard deviation. Data

were analysed using two-tailed Student´s t test (P < 0.001).

***

***

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

flu

ore

scen

ce n

ucle

us/c

yto

so

l

Col-0 4xaly thp1

C

Col-0 4xaly thp1

B

+Yra1

+ALY1

+ALY1

+ALY1

empty

vector

A



Parsed CitationsAntosch M, Schubert V, Holzinger P, Houben A, Grasser KD. (2015) Mitotic lifecycle of chromosomal 3xHMG-box proteins and the roleof their N-terminal domain in the association with rDNA loci and proteolysis. New Phytol 208:1067-1077

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Antosz W, Pfab A, Ehrnsberger HF, Holzinger P, Köllen K, Mortensen SA, Bruckmann A, Schubert T, Längst G, Griesenbeck J,Schubert V, Grasser M, Grasser KD. (2017) Composition of the Arabidopsis RNA polymerase II transcript elongation complex revealsthe interplay between elongation and mRNA processing factors. Plant Cell 29:854-870


Bruhn L, Munnerlyn A, Grosschedl R. (1997) ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalphaenhancer function. Genes Dev 11:640-653


Burd CG, Dreyfuss G. (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615-621Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Canto T, Uhrig JF, Swanson M, Wright KM, MacFarlane SA. (2006) Translocation of tomato bushy stunt virus P19 protein into thenucleus by ALY proteins compromises its silencing suppressor activity. J Virol 80:9064-9072


Chen MQ, Zhang AH, Zhang Q, Zhang BC, Nan J, Li X, Liu N, Qu H, Lu CM, Sudmorgen, Zhou YH, Xu ZH, Bai SN. (2012) ArabidopsisNMD3 is required for nuclear export of 60S ribosomal subunits and affects secondary cell wall thickening. PLoS One 7:e35904


David R, Burgess A, Parker B, Li J, Pulsford K, Sibbritt T, Preiss T, Searle IR. (2017) Transcriptome-wide mapping of RNA 5-methylcytosine in Arabidopsis mRNAs and noncoding RNAs. Plant Cell 29:445-460


Dufu K, Livingstone MJ, Seebacher J, Gygi SP, Wilson SA, Reed R. (2010) ATP is required for interactions between UAP56 and twoconserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex. Genes Dev 24:2043-2053


Dürr J, Lolas IB, Sørensen BB, Schubert V, Houben A, Melzer M, Deutzmann R, Grasser M, Grasser KD. (2014) The transcriptelongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis. Nucleic Acids Res 42:4332-4347


Francis KE, Lam SY, Copenhaver GP. (2006) Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectinmethylesterase gene. Plant Physiol 142:1004-1013


Gaouar O, Germain H. (2013) mRNA export: threading the needle. Front Plant Sci 4:59Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gatfield D, Izaurralde E. (2002) REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. JCell Biol 159:579-588


Germain H, Qu N, Cheng YT, Lee E, Huang Y, Dong OX, Gannon P, Huang S, Ding P, Li Y, Sack F, Zhang Y, Li X. (2010) MOS11: a new www.plantphysiol.orgon October 19, 2020 - Published by Downloaded from

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