The nuclear pore protein AtTPR is required for RNA homeostasis, flowering time, and auxin signaling

Abstract

Nuclear pore complexes (NPCs) mediate the transport of RNA and other cargo between the nucleus and the cytoplasm. In vertebrates, the NPC protein TRANSLOCATED PROMOTER REGION (TPR) is associated with the inner filaments of the nuclear basket and is thought to serve as a scaffold for the assembly of transport machinery. In a screen for mutants that suppress the expression of the floral inhibitor FLOWERING LOCUS C, we identified lesions in the Arabidopsis (Arabidopsis thaliana) homolog of TPR (AtTPR). attpr mutants exhibit early-flowering and other pleiotropic phenotypes. A possible explanation for these developmental defects is that attpr mutants exhibit an approximately 8-fold increase in nuclear polyA RNA. Thus AtTPR is required for the efficient export of RNA from the nucleus. Microarray analysis shows that, in wild type, transcript abundance in the nuclear and total RNA pools are highly correlated; whereas, in attpr mutants, a significantly larger fraction of transcripts is enriched in either the nuclear or total pool. Thus AtTPR is required for homeostasis between nuclear and cytoplasmic RNA. We also show that the effects of AtTPR on small RNA abundance and auxin signaling are similar to that of two other NPC-associated proteins, HASTY (HST) and SUPPRESSOR OF AUXIN RESISTANCE3 (SAR3). This suggests that AtTPR, HST, and SAR3 may play related roles in the function of the nuclear pore.

Figure 1.

attpr mutations and their effect on flowering time. ld-1 (A) and lds30B (B) grown in long days for 4 weeks. C, The effect of attpr mutations on flowering time in the indicated backgrounds. Black and gray bars represent the number of primary rosette leaves formed prior to flowering in long and short days. The white portions of the bars represent the number of cauline leaves. Error bars indicate one sd. D, A schematic representation of AtTPR; the thin line indicates the chromosome, thick lines indicate exons. The positions of T-DNA insertions in attpr mutants are indicated above the gene. [See online article for color version of this figure.]

Figure 2.

Effect of attpr on development and gene expression. A, Rosette leaves formed prior to flowering in long days by Col (top) and attpr (bottom). B to G, Scanning electron micrographs of Col (B, D, and F) and attpr (C, E, and G) inflorescences (B and C) and sepals (D–G). White bars represent 1 mm (B and C), 100 μm (D and E), and 10 μm (F and G). H, RT-PCR analysis of gene expression in the indicated genotypes; numbers indicate fold changes relative to Col. RNA was isolated from 2-week-old seedlings grown under long days. UBQ10 was used as a constitutively expressed control. [See online article for color version of this figure.]

Figure 3.

AtTPR expression. A, RT-PCR analysis of gene expression in the indicated genotypes and tissues. UBQ10 was used as a constitutively expressed control. B to E, Histochemical expression of GUS expression in AtTPR∷GUS transgenic plants.

Figure 4.

RNA analysis in attpr mutants. A to C, Whole-mount in situ localization of polyA RNA. Confocal images of leaves of Col (A and B) and attpr (C) hybridized without (A) or with (B and C) a poly(dT) fluorescein-tagged oligonucleotide. Leaves were harvested from 3-week-old plants grown under short days (A–C). D and E, RNA-blot analysis of polyA RNA in nuclear and total RNA from Col and attpr. Blots were probed with a radiolabeled poly(dT) oligonucleotide. D, Numbers indicate fold changes relative to Col. E, Size distribution of polyA RNA in Col and attpr total RNA. Photographs of the ethidium-stained gels used to produce the RNA blots are shown in the bottom sections (D and E) and were used as a control for loading. F to I, Microarray analysis in Col and the attpr mutant. Scatter plots of normalized signal intensities are shown for the indicated samples.

Figure 5.

Effects of attpr on small RNA abundance and auxin signaling. A, RNA-blot analysis of small RNAs. Total RNA was isolated from the above-ground portions of long-day-grown flowering plants. B, Root elongation after 4 d on auxin-containing media for the indicated genotypes. C, Effect of sar3 on flowering time in a FRI-containing background. B and C, Error bars indicate one sd.