The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization.
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.
Tam
Patrick P C
PP
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada.
Barrette-Ng
Isabelle H
IH
Simon
Dawn M
DM
Tam
Michael W C
MW
Ang
Amanda L
AL
Muench
Douglas G
DG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D055028:Comparative Genomic Hybridization; D024202:Electrophoretic Mobility Shift Assay; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010940:Plant Proteins; D016601:RNA-Binding Proteins