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

Abstract

BackgroundPuf 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.ResultsThe 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.ConclusionsThe 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.

Pumilio Puf domain RNA-binding proteins in Arabidopsis

“…Any mutation in the ribosomal subunit genes affects auxin biosynthesis due to defective ribosomes, which translationally repress the auxin responsive factors, such cellular localization demonstrated that cytoplasmic punctuate structures are formed for many of the Arabidopsis Puf proteins, such as APUM7 (At1g78160), APUM8 (At1g22240), APUM9, APUM10, APUM12 (At5g56510), APUM14 (At5g43110) and APUM18. 44 However, others, i.e., APUM23 (At1g72320) and APUM24 (At3g16810), localize to the nucleolus, 44,45 suggesting that certain Puf proteins are involved in organelle-specific functions.…”

ArabidopsisPumilio protein APUM5 suppressesCucumber mosaic virusinfection via direct binding of viral RNAs

“…The outer surface of the domain permits protein-protein interactions with diverse proteins, such as deadenylase and general translation factors (9, 10). Arabidopsis Pufs also exhibit RNA-binding activity and have conserved binding motifs (11,12). However, plant Puf functions have not yet been fully identified or characterized.…”

Plant defense responses against viral and bacterial pathogen infections. Focus on RNA-binding proteins (RBPs)

“…These two types of domains have been also found in a broad spectrum of other species ranging from bacteria to humans, what suggests that they might be ancient protein structures [29]. Other common domains found in RBPs are zinc finger domain (ZnF, mostly C-x8-C-x5-C-x3-H type) [32,33], cold-shock domain, DEAD/DEAH box [34], Pumilio/FBF (PUF) domain, double-stranded RNA binding domain (RS-RBD) [35], and Piwi/Argonaute/Zwille (PAZ) domain [36]. A single RBP may possess multiple binding domains [33][34][35].…”

“…Other common domains found in RBPs are zinc finger domain (ZnF, mostly C-x8-C-x5-C-x3-H type) [32,33], cold-shock domain, DEAD/DEAH box [34], Pumilio/FBF (PUF) domain, double-stranded RNA binding domain (RS-RBD) [35], and Piwi/Argonaute/Zwille (PAZ) domain [36]. A single RBP may possess multiple binding domains [33][34][35]. Moreover, auxiliary motifs, such as arginine-rich, glycinerich, arginine-glycine (RGG) or serine-arginine (SR) repeats are often present in RBPs ( fig.…”