Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

Rory F Degenhardt,Peta C Bonham-Smith

doi:10.1104/pp.107.111799

Abstract

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability.

Highlights

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins)
It remains to be determined whether these findings result from a biological shortage of r-proteins for ribosome biogenesis caused by the loss of one copy from a redundant family, or if they are indicative of specialized functions for specific paralogs
We found that only RPL23aA is able to tolerate N-terminal GFP5 fusions and C-terminal fusion to monomeric red fluorescent protein (mRFP) without disrupting localization

Summary

Introduction

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). The Arabidopsis genome contains 254 genes for the 81 r-proteins, with families of between two and five expressed members (Barakat et al, 2001; Chang et al, 2005) This high degree of paralogy suggests that many r-protein genes may be functionally redundant or development/tissue/stress specific. In Arabidopsis, rps13b and rps18a T-DNA insertional mutants develop narrow, pointed first leaves and have stunted root growth and delayed flowering; in both cases, northern analyses confirmed that transcript levels from paralogs were unaffected (Van Lijsebettens et al, 1994; Ito et al, 2000) It remains to be determined whether these findings result from a biological shortage of r-proteins for ribosome biogenesis caused by the loss of one copy from a redundant family, or if they are indicative of specialized functions for specific paralogs

Methods

Results

Conclusion