Recombinant Arabidopsis thaliana Exportin-T (PSD), partial

What is the function of PSD (PAUSED) in Arabidopsis thaliana?

PSD is the Arabidopsis ortholog of exportin-t (XPOT), a protein that mediates the nuclear export of tRNAs. This protein contains a conserved Ran-binding domain at the N-terminus, which is a signature of the importin-β protein family. PSD functions in nuclear-cytoplasmic transport pathways, specifically facilitating the movement of tRNAs from the nucleus to the cytoplasm in a Ran-dependent manner. Research has demonstrated that PSD is capable of rescuing the tRNA export defect of los1 mutants in Saccharomyces cerevisiae, suggesting that its function in tRNA transport has been evolutionarily conserved .

How does PSD in Arabidopsis compare to its homologs in other organisms?

The PSD protein in Arabidopsis shares 27% identity and 48% similarity with human exportin-t, and 21% identity and 41% similarity with yeast Los1p. Interestingly, PSD and human exportin-t are more closely related to each other than either is to yeast Los1p. All three proteins contain a conserved Ran-binding domain characteristic of the importin-β family. Despite these similarities, there are functional differences between these proteins. Unlike yeast and mammals, where mutations in exportin-t show specific phenotypes, mutations in Arabidopsis PSD result in pleiotropic developmental defects, suggesting a broader role in plant development .

How is the PSD gene expressed in Arabidopsis?

Northern blot analyses have revealed that PSD is expressed as a single transcript of approximately 4 kb throughout Arabidopsis development. The expression level is relatively low but detectable in roots, vegetative leaves, and floral buds. Semi-quantitative RT-PCR analysis has shown that PSD is expressed at similar levels in the shoot apex of plants at different developmental stages (8, 15, or 22 days under short-day conditions). The 3'-UTR of PSD undergoes alternative splicing, with variations in exons 13 and 14. This widespread expression pattern is consistent with PSD's pleiotropic role in development .

What are the recommended storage conditions for recombinant Arabidopsis thaliana Exportin-T (PSD)?

For optimal stability of recombinant PSD protein, the recommended storage conditions depend on the preparation form. For liquid formulations, the shelf life is typically 6 months when stored at -20°C/-80°C. Lyophilized forms can be stored for up to 12 months at -20°C/-80°C. Working aliquots should be stored at 4°C and used within one week. Repeated freezing and thawing cycles should be avoided as they can compromise protein stability and function .

How should recombinant PSD protein be reconstituted for experimental use?

For reconstitution of lyophilized recombinant PSD, it is recommended to briefly centrifuge the vial before opening to bring the contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage of the reconstituted protein, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and to aliquot the solution before storing at -20°C/-80°C .

What experimental approaches can be used to study PSD function in Arabidopsis?

Several experimental approaches have been effectively used to study PSD function:

• Genetic analysis: Utilizing T-DNA insertion mutants (psd alleles) to study loss-of-function phenotypes.

• Complementation studies: Expressing PSD in yeast los1 mutants to verify functional conservation.

• RNA analysis: Northern blotting and RT-PCR to examine PSD expression patterns.

• tRNA processing analysis: Examining the effects of psd mutations on tRNA splicing and nuclear export.

• Double mutant analysis: Creating psd;hst double mutants to study potential redundancy in RNA export pathways.

• Developmental phenotyping: Detailed characterization of developmental defects in various tissues and at different growth stages .

What developmental processes are affected by mutations in the PSD gene?

Mutations in PSD affect multiple developmental processes in Arabidopsis, indicating its pleiotropic role. The key developmental defects observed include:

• Disruption in the initiation of the shoot apical meristem

• Delay in leaf initiation after germination

• Delayed emergence of the radicle and lateral roots

• Delay in the transition to flowering

These phenotypes suggest that PSD plays a critical role in major developmental transitions in Arabidopsis. In the hua1-1 hua2-1 background, psd mutations also cause transformation of reproductive organs into perianth organs, resembling mutations in the floral homeotic gene AGAMOUS .

How do PSD mutations affect tRNA processing and export in Arabidopsis?

In Arabidopsis, PSD mutations interfere with tRNA processing, particularly affecting tRNA-Tyr. Analysis of nuclear and cytoplasmic fractions from psd-13 mutants shows an increased level of unspliced tRNA-Tyr in the nucleus and decreased levels of spliced tRNA-Tyr in the cytoplasm. There is also a slight increase in intron-less tRNA-Met in the nucleus, though the cytoplasmic levels remain unaffected. This suggests that PSD plays a specific role in the export of certain tRNAs, particularly those that require splicing .

What is the relationship between PSD and HST (HASTY) in RNA export pathways?

PSD and HST (the Arabidopsis ortholog of exportin 5) function in separate RNA export pathways:

• HST mutations reduce the accumulation of most miRNAs but do not affect tRNA accumulation.

• PSD mutations affect tRNA-Tyr processing but do not impact miRNA accumulation or nuclear export.

• Double mutants (psd;hst) show a more severe phenotype than either single mutant, combining characteristics of both: accelerated trichome production and upwardly curled leaves (typical of hst) with delayed leaf initiation (typical of psd).

These findings indicate that HST and PSD do not share RNA cargoes for nuclear export, and multiple nuclear export pathways exist for these small RNAs in Arabidopsis .

What redundant tRNA export pathways exist in Arabidopsis?

Despite the role of PSD in tRNA export, null mutations in PSD are viable, indicating the existence of redundant tRNA export pathways in Arabidopsis. Several potential alternate pathways have been proposed:

• HST/Exportin-5 pathway: Though HST (Arabidopsis ortholog of exportin 5) has been shown to bind tRNAs in mammals, psd;hst double mutants remain viable, suggesting additional pathways.

• Passive diffusion: tRNAs might pass through nuclear pores via passive diffusion at sufficient rates for survival.

• Unknown exportins: Other members of the importin-β family might contribute to tRNA export.

• Nuclear aminoacylation pathway: Similar to yeast, Arabidopsis might have a pathway requiring nuclear aminoacylation of tRNAs before export.

The viability of psd;hst double mutants suggests that Arabidopsis has more than two tRNA export pathways, similar to the model proposed for yeast .

How does the nuclear pore complex (NPC) in Arabidopsis relate to PSD function?

The nuclear pore complex (NPC) in Arabidopsis contains at least 30 nucleoporins, through which PSD mediates tRNA export. Interestingly, plant nucleoporins exhibit higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins, despite plants and yeast both being viable with mutations in tRNA exportins. The plant NPC lacks seven components present in vertebrate NPCs but possesses a unique nucleoporin, Nup136/Nup1, that contains Phe-Gly repeats. This distinctive NPC composition may impact how PSD functions in nuclear export. Research into Arabidopsis NPC components has provided valuable insights into the structure and function of plant nuclear transport systems, including those involving PSD .

What are the molecular mechanisms underlying the developmental defects in psd mutants?

• Spatial regulation: Although PSD is widely expressed, it may be subject to tissue- or cell-specific transcriptional regulation.

• Translational control: The alternative splicing of the PSD 3'-UTR may affect its translation in response to environmental factors or developmental cues.

• Developmental sensitivity: Major developmental transitions (meristem initiation, lateral root emergence, flowering) may have higher translational demands, making them particularly vulnerable to tRNA export defects.

• tRNA specificity: Some tRNAs may be more dependent on PSD for export than others, affecting the translation of specific proteins .

What are the key considerations when working with recombinant Arabidopsis thaliana Exportin-T (PSD)?

When working with recombinant PSD protein, researchers should consider:

• Source: Recombinant PSD can be produced in E. coli or mammalian cell systems, with potential differences in post-translational modifications and activity.

• Purity: Commercially available recombinant PSD typically has >85% purity as determined by SDS-PAGE.

• Stability: The protein should be stored appropriately to maintain activity (see storage recommendations).

• Functional assays: When using recombinant PSD for functional studies, it's important to verify its activity, potentially through complementation assays in yeast los1 mutants.

• Protein length: Note that commercially available recombinant PSD is often partial, not the full-length protein, which may affect certain functional studies .

How can researchers effectively study the interaction between PSD and the Ran GTPase cycle?

To study the interaction between PSD and the Ran GTPase cycle:

• In vitro binding assays: Using purified recombinant PSD and Ran-GTP to assess direct binding.

• Co-immunoprecipitation: To detect PSD-Ran interactions in plant cell extracts.

• Yeast two-hybrid analysis: Similar to the approach used with HST, which was shown to interact with Ran.

• Site-directed mutagenesis: Targeting the conserved Ran-binding domain at the N-terminus of PSD to assess the functional importance of this interaction.

• Fluorescence microscopy: Using fluorescently tagged proteins to visualize interactions in vivo.

• Ran mutant analysis: Utilizing Ran mutants locked in GTP- or GDP-bound states to study the directionality of transport .

What experimental design would be optimal for analyzing potential additional cargoes of PSD beyond tRNAs?

To identify potential additional cargoes of PSD beyond tRNAs, an optimal experimental design might include:

• Immunoprecipitation-RNA analysis: Immunoprecipitating PSD and analyzing associated RNAs through RNA-seq or other high-throughput methods.

• CLIP-seq (Cross-linking immunoprecipitation-sequencing): To identify direct RNA binding sites of PSD.

• Comparative RNA-seq: Analyzing nuclear vs. cytoplasmic RNA ratios in wild-type vs. psd mutant plants to identify RNAs that accumulate in the nucleus in the absence of PSD.

• In vitro binding assays: Testing the ability of recombinant PSD to bind different RNA species.

• Genetic screens: Identifying suppressors or enhancers of psd phenotypes that might reveal genes involved in alternative cargo transport.

• Differential gene expression analysis: Comparing expression patterns in psd single mutants vs. psd;hst double mutants to identify uniquely affected transcripts .