NUA Activities at the Plant Nuclear Pore

Abstract

NUA (Nuclear Pore Anchor), the Arabidopsis homolog of Tpr (Translocated Promoter Region), is one of the few nuclear pore proteins conserved between animals, yeast and plants. In the May issue of Plant Cell, we report that null mutants of NUA show a pleiotropic, early flowering phenotype accompanied by changes in SUMo and RNA homeostasis. We have shown that the early flowering phenotype is caused by changed abundances of flowering time regulators involved in several pathways. Arabidopsis nua mutants phenocopy mutants lacking the ESD4 (EARlY IN ShoRT DAYS 4) SUMo protease, similar to mutants of their respective yeast homologs. however, in contrast to the comparable yeast mutants, ESD4 does not appear to be delocalized from the nuclear pore in nua mutants. Taken together, our experimental data suggests a role for NUA in controlling mRNA export from the nucleus as well as SUMo protease activity at the nuclear pore, comparable but not identical to its homologs in other eukaryotes. Furthermore, characterization of NUA illustrates a potential link at the nuclear pore between SUMo modification, RNA homeostasis and plant developmental control.

Introduction

The nuclear pore complex (NPC) is essential for all macromolecule exchange between the nucleus and the cytoplasm. Around 30 nucleoporins (Nups), the protein components of the NPC, have been identified and studied extensively in yeast and animal systems.^1–3 In contrast, information is still limited about the plant NPC. Recently, putative plant Nups were identified by genetic screens for signal transduction components involved in plant-microbe interactions, auxin signaling and cold stress tolerance.^4–8 The diverse effects of mutations in these plant Nups underline that the communication through the NPC is involved in many cellular and developmental processes.

Nuclear-Pore Anchor (NUA), The Arabidopsis Homolog of tpr/Mlp1/Mlp2/Megator

Vertebrate Tpr (Translocated promoter region) and its yeast homologs Mlp1/Mlp2 (Myosin-Like Protein 1/2), as well as the Drosophila homolog Megator, are long coiled-coil proteins associated with the nuclear basket of the NPC.^9–11 They are involved in mRNA export, unspliced RNA retention, telomere organization, spindle pole assembly, docking of SUMO proteases and spatially regulated gene expression.

We have recently identified NUA, a single Arabidopsis gene encoding a ca. 220kD protein, as the homolog of Tpr/Mlp1/Mlp2/Megator.¹² Immunolocalization in Arabidopsis root cells demonstrates that NUA is located at the inner surface of the nuclear envelope (NE) during interphase and in the vicinity of the spindle during prometaphase. The later observation suggests a potential role for NUA in spindle matrix organization as being reported for its Drosophila homolog.¹³ Consistent with mammalian Tpr being only peripherally attached to the NPC and recruited to the reassembled NPC relatively late during telophase, NPC assembly and recruitment of other plant nucleoporins like WPP-domain Interacting Protein 1 (WIP1) precedes the NPC association of NUA (Fig. 1A).^14,15

Figure 1.

(A) Immunofluorescence in GFP-WIP1 transgenic Arabidopsis root tips with anti-NUA and anti-GFP antibodies. DNA was stained with DAPI. The arrow indicates the reforming daughter NE; the arrowhead points to the cell plate. Plant material, antibodies, immunolabeling and microscopy were performed as described (ref. ¹²). (B) Immunofluorescence in Arabidopsis root tips with anti-NUA and NE marker QE5 antibodies. Bottom panels, Fluorescence profiles of the white dash lines in overlay images. The seemingly higher cytoplasmic signal from anti-NUA seen in nua-2 and nua-3 likely is non-specific. Images of nua-2 and nua-3 were taken at higher light sensitivity to show the much weaker immunofluorescence decoration on the NE in nua-2 and the complete absence of NE decoration in nua-3. Plant material, antibodies, immunolabeling and microscopy were performed as described (ref. ¹²). All scale bars: 10 µm. (C) Model of NUA action at the nuclear pore. Both NUA and ESD4 are located at the nuclear periphery, where they likely interact. Absence of NUA or of ESD4 inhibits desumoylation. Absence of NUA inhibits mRNA export and miRNA homeostasis and affects expression levels of key regulatory genes. We propose that NUA links SUMo and RNA homeostasis and gene expression at the nuclear pore, thereby affecting several molecular processes required for proper plant development.

Knock-Out and Knock-Down Mutant Phenotypes of Arabidopsis NUA

We characterized four T-DNA insertion mutants for NUA in detail. In the order of nua-2, nua-3 and nua-1/nua-4, they comprise an allelic series of increasing severity for several correlating phenotypes. These include early flowering under short days and long days, increased abundance of SUMO (small ubiquitin-related modifier) conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA.

The knock-out mutants nua-1 and nua-4 have additional pleiotropic developmental phenotypes, including stunted growth, defects in stamen and silique development, and changes in phyllotaxy. The extreme early flowering phenotype of nua-1/4 mutants is consistent with molecular changes in several flowering pathways, such as the reduction of FLOWERINg LOCUS C (FLC) expression and a concomitant increase of FLOWERINg LOCUS T (FT) and SUPPRESSOR OF OVERExPRESSION OF CO 1 (SOC1) expression, a reduction of MADS AFFECTINg FLOWERINg 4 (MAF4) expression, as well as an increase of MYB33, MYB65 and LEAFY (LFY). Since MYB33 and MYB65 gene expression is post-transcriptionally regulated by miR159, their upregulation is consistent with the recent report of a reduction of miR159 in a nua mutant.^16,17 However, it still remains undetermined whether NUA is involved in miRNA export, miRNA processing, or both.

The finding that nua-3 acts as a functional knockdown of intermediate severity, is intriguing. We hypothesize that the truncated N-terminal partial protein expressed in nua-3 might possess part of the NUA activity involved in RNA and SUMO homeostasis as well as developmental regulation. Interestingly, this fragment overlaps with the interaction domain binding to the SUMO protease EARLY IN SHORT DAYS 4 (ESD4) as determined in yeast two-hybrid assays.^18,19 Although the ∼100kD partial protein in nua-3 can be detected by immunoprecipitation and appears more abundant than the NUA protein present in nua-2, immunofluorescence decoration of the NE/NPC can only be seen in nua-2 but not in nua-3 (Fig. 1B). This would be consistent with the loss of putative nuclear localization signals in the missing C-terminal tail and appears to indicate that nuclear pore association is not required for partial function of the nua-3 fragment.

Connection between Sumoylation, mRNA Export and Plant Developmental Control

Both on whole-plant and on molecular level, nua-1/4 mutants phenocopy mutants of ESD4, an Arabidopsis SUMO protease concentrated around the nuclear periphery.^18,19 We found that NUA and ESD4 interact in yeast two-hybrid assays and nua-1 esd4-2 double mutants resemble nua-1 and esd4-2 single mutants, suggesting that the two proteins may act in a shared pathway or complex. In yeast, Mlp1 and Mlp2 function in anchoring the ESD4 homolog Ubl-specific Protease 1 (Ulp1) to the NPC and this attachment is crucial for Ulp1 function.²⁰ However, analyzing gFP-ESD4 protein location in nua mutants shows no delocalization of ESD4, suggesting a different functional connection between NUA and ESD4.

In yeast and mammals, the identification of hnRNPs and RNA helicases as substrates for SUMO modification suggested a link between sumoylation and RNA metabolism.^21–23 In plants, NUA provides the first link between sumoylation, mRNA export and plant developmental regulation. However, how these three processes are connected molecularly and mechanistically remains elusive and an exciting direction to be pursued. Several Arabidopsis mutants are available in which either mRNA export is defective, like los4, or SUMO homeostasis is disrupted, such as esd4, siz1 and the SUMO1/2 overexpressor.^19,24–26 It will be of great interest to investigate any correlation between SUMO and RNA homeostasis in these mutants.

Among all the developmental defects in the nua mutants, flowering time regulation was analyzed in the greatest detail. One of the key factors misregulated in nua mutants is FLC, whose expression is controlled via epigenetic silencing and chromatin remodeling in the autonomous pathway. Several autonomous pathway components are involved in RNA binding and RNA processing.²⁷ Our analysis of nua mutants further strengthens the point that RNA metabolism is important for flowering time regulation. Perhaps, a more intriguing speculation is that NUA, like its yeast homologs Mlp1/Mlp2 (ref. ²⁸) may be involved in the nuclear architecture and spatial positioning of the genome which affects the transcriptional state of key regulators and/or more directly the epigenetic control of FLC expression.

Conclusions and Perspectives

The pleiotropic developmental and molecular phenotypes of nua mutants are the first demonstration of the effects of a null mutant of a Tpr-like protein on a multi-cellular whole-organism level. Our data suggest that the long coiled-coil protein NUA is important for the spatial organization and regulation of mRNA export and sumoylation in plants, and that disruption in these processes affects the signaling events involved in diverse developmental processes (Fig. 1C). Because of the role of coiled-coil domains in scaffolding via protein-protein interactions, it will be essential to isolate in vivo binding partners for NUA to fully understand its function on the molecular level. Proteins involved in mRNA export and targets of sumoylation are conceivable interacting proteins for NUA.

Addendum to: Xu XM, Rose A, Muthuswamy S, Jeong SY, Venkatakrishnan S, Zhao Q, Meier I. NUCLEAR PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development. Plant Cell. 2007;19:1537–1548. doi: 10.1105/tpc.106.049239.