Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

Abstract

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.

Figure 1.

Preparation of Arabidopsis nucleoli and classification of proteins identified by mass spectrometry. (A) Nucleoli were purified from protoplasts and visualized using phase contrast light microscopy. Left: purified protoplasts (bar, 10 μm); center: purified nuclei (bar, 5 μm); right: purified nucleoli (bar, 2 μm). (B) Distribution of identified proteins among different protein classes

Figure 2.

GFP localizations of a selection of proteins identified in the Arabidopsis nucleolar proteomic analysis. Confocal GFP images of single cells are shown and nucleoli indicated by arrows. (A-D) Ribosomal proteins: RPL4.1 (At5g02870), RPL9 (At1g33120), RPL3 (At1g43170), and RPL10A (At5g22440). Note that some ribosomal protein fusions label the cytoplasm strongly, others weakly; (E-H snoRNP and snRNP proteins: GAR1 (At3g03920), U3-55k (At4g05410), SmB (At5g44500), and SmD3 (At1g76300); (I-L) DNA-interacting proteins: H2A (At1g51060), H2A (At5g27670), H1 (At2g30620), and Zinc-finger protein (At5g03740). Note that I and J are both H2A histones, but the homologue in I is widely present throughout the chromatin, whereas that in J is apparently concentrated in heterochromatin; (M-P) RNA-interacting proteins: glycine-rich RNA-binding protein (At4g39260), DDX18 (At3g18600), hnRNPA1 (At3g15010), and PRP19 (At1g04510); (Q-T) proteins with unknown function: plant-specific proteins (At4g25210 and At5g64680), and conserved proteins in both plant and human proteomes (At3g56990 and At3g58660). Bar, 5 μm.

Figure 3.

GFP localizations of EJC proteins identified in the Arabidopsis nucleolar proteomic analysis. Images of three different cells are shown for each of the Y14, Mago, RNPS1, UAP56-2, ALY-4, and eIF4A-III GFP fusions. A single z plane passing through the center of a nucleolus from a deconvoluted CCD image is shown in each case. Bar, 5 μm.

Figure 4.

GFP localizations of the three ALY homologues not detected in the Arabidopsis nucleolar proteomic analysis. Images of three different cells are shown for ALY-1, ALY-2, and ALY-3 GFP fusions. A single z plane passing through the center of a nucleolus from a deconvoluted CCD image is shown in each case. Bar, 5 μm.