The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

Abstract

Background: Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.

Results: The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.

Conclusions: The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

Figure 1

A maximum likelihood phylogenetic tree of the PUM-HDs of Arabidopsis, rice and other plant and non-plant species. The analysis is based on the deduced amino acid sequence of the PUM-HD domain from each predicted Puf gene. The tree includes all members from Arabidopsis and rice, and representative members from Physcomitrella patens (Phys), Chlamydomonas reinhardii (Chlamy), Saccharomyces cerevisiae (Sc), as well as Drosophila Pumilio (DrPumilio) and human Pum1 (HsPum1). The Arabidopsis genes are referred by their designated Pum gene number (i.e., AtPumxx) that were reported by the National Center for Biotechnology Information (NCBI), as well as their gene locus name (Atxxxxxxx). The rice clones are identified by their gene locus name only (Osxxxxxxxx), as standardized Pum gene designations have not yet been established. Maximum likelihood bootstrap values (>65%) are shown above the nodes (PhyML/RaxML), and Bayesian posterior probability values (>0.95) are shown below the nodes. The bar at the bottom of the figure indicates the number of substitutions per site. The tree is rooted at its midpoint and, thus, its rooting should be interpreted as an hypothesis.

Figure 2

Schematic line diagram comparing the primary structure of Puf proteins in Arabidopsis and rice. The numbered Puf repeats in the PUM-HD of each protein are indicated (alternating black and yellow strips), and the 1' and 8' pseudorepeats are also identified (blue). A conserved nucleic acid binding protein domain (NABP) is present in several Arabidopsis and rice PUM-HDs (red). Three additional Puf repeats were identified outside of the PUM-HD in AtPum23 (green). Two versions of the 'domain of unknown function' (DUF) were identified in Os08g40830 (green). The length of each protein is indicated in parentheses. Sequences that are supported by cDNA sequences are identified (*). The AtPum13 and AtPum22 cDNAs were amplified and sequenced independently (PPC Tam and DG Muench, unpublished observations).

Figure 3

Amino acid sequence alignment of the PUM-HD encoded by Puf genes in various organisms. Arabidopsis thaliana (AtPum2); Oryza sativa (Os01g62650); Physcomitrella patens (PpPum1, AAX58753); Chlamydomonas reinhardii (CrPuf, XP001703567); Drosophila melanogaster (DmPumilio); Homo sapiens (HsPUM1);Caenorhabditis elegans (CePuf9); and Saccharomyces cerevisiae (ScPuf3p). Identical amino acids are marked in black and similar residues are marked in gray.

Figure 4

Alignment of amino acids in the PUM-HD that are predicted to interact with RNA bases. Sequence alignment of amino acid triplets at positions 12, 13 and 16 in each Puf repeat (R1 to R8) from the Arabidopsis and rice Puf proteins. Black shading identifies amino acids that are identical to the human Pum1 protein.

Figure 5

Models of the plant PUM-HD bound to RNA. (A, B) Ribbon (left) and stick (right) models of the PUM-HDs of AtPUM2 (A) and Os01g62650 (B) bound to the RNA bases of Box 2 of the NRE (UUGUAUAU) that interact with Puf repeats 2 to 8. The RNA is shown as a ball-and-stick model. In the ribbon diagrams, the amino acid side chains that interact with the Watson-Crick edge of each base are shown in green, and those that provide potential stacking interactions are colored magenta. In the stick models, only the amino acid side chains that contact RNA bases are shown. The extended loop between repeat 7 and 8 is identified (*). (C, D) Sequence alignment of residues in helix 2 of repeats 1-8 that provide putative RNA contact sites on the concave surface of the PUM-HD of AtPum2 (C) and Os01g62650 (D). Numbers above the sequences represent the position of each amino acid each Puf repeat. Numbers in brackets refer to the position of the first amino acid in the complete AtPum2 and Os01g62650 polypeptide sequence. Boxes surround the amino acid residues at positions 12, 13 and 16. (E, F) Schematic diagram showing the protein:RNA contacts in the models of the AtPum2 (E) and Os01g62650 (F) PUM-HDs bound to the NRE1. Dotted lines indicate potential hydrogen bonds, dashed lines indicate potential stacking interactions, and ')))))' indicates potential van der Waals interactions. Distances between atoms indicated on the lines are indicated in Ångstroms.

Figure 6

Models of the AtPum13 PUM-HD bound to RNA. Ribbon (A) and stick (B) models of the PUM-HD of AtPUM13 bound to the core nucleotides of Box 2 of the NRE1 (UUGUAUAU). (C) Sequence alignment of residues in helix 2 of repeats 1-8 that provide putative RNA contact sites on the concave surface of the PUM-HD. (D) Schematic diagram showing the protein:RNA contacts in the model of the AtPum13 PUM-HD. Legend details are described in Figure 5.

Figure 7

AtPUM-HD-2 demonstrates binding specificity to wildtype NRE1. EMSAs using purified GST-AtPum2 PUM-HD and wildtype or point mutated NRE1. (A) EMSA assays using the wildtype (NRE) or mutant (G to U) NRE1 RNA oligonucleotide in the absence or presence of GST or GST-AtPum2 PUM-HD (PUM). Unbound radiolabelled RNA (Free) shifts to a high molecular weight complex when bound to GST-AtPum2 PUM-HD (Bound). The fraction of bound RNA (B_f) was determined for each reaction. 100-fold excess non-labelled mutant NRE1 was added to the reaction containing labelled WT-NRE (NRE + comp). Conversely, 100-fold excess of non-labelled wildtype NRE1 was added to the reaction containing labelled mutant NRE RNA (G to U + comp). The underlined RNA sequence corresponds to cognate RNA that interacts with repeats 1 through 8 in the Drosophila Pumilio PUM-HD. (B) EMSA titration of WT-NRE1 and increasing concentrations of GST-AtPum2 PUM-HD (PUM-HD). The protein concentrations were 0, 0.12, 0.25, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 nM. (C) The fraction of bound WT-RNA as a function of GST-AtPum2 PUM-HD from the EMSA in (B) was plotted and the dissociation constant (K_d) was determined. (D) EMSA titration of mutant NRE1 (G to U) and increasing concentrations of GST-AtPum2 PUM-HD. The protein concentrations were 0, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 nM.

Figure 8

Subcellular localization of AtPum proteins. Representative epifluorescence images of cells expressing AtPum proteins fused to the amino terminus of GFP or RFP in onion (A to D) or fava bean (E to I) epidermal cells. (A) AtPum7-GFP, (B) AtPum8-RFP, (C) AtPum9-RFP, (D) AtPum10-GFP, (E) AtPum12-GFP, (F) AtPum14-GFP, (G) AtPum18-RFP, (H) AtPum23-GFP, and (I) AtPum24-GFP. Arrow identifies heavily stained region that likely resulted from aggregation of protein complexes. Bar, 10 microns.

Figure 9

Leptomycin B treatment results in an enrichment of AtPum18-RFP, but not AtPum10-GFP, in the nucleus. Bar, 10 microns.

Figure 10

AtPum23 and AtPum24 co-localize with the nucleolar marker RFP-PRH75. Bar, 1 micron.