Arabidopsis L10 ribosomal proteins in UV-B responses

Abstract

Ribosomal protein L10 (RPL10) is a ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; however, increasing evidence indicates that RPL10 from various organisms has multiple extra-ribosomal functions, besides being a constituent of ribosome and its role in translation. Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C. Previously, we found that in maize and in A. thaliana, UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B. Here, we show that RPL10s are differentially regulated by UV-B in a dosage and time dependent manner: RPL10C is induced, RPL10B is downregulated at high UV-B intensity and RPL10A is not UV-B regulated. In addition, by co-immunoprecipitation studies using RPL10 antibodies and proteins from control and UV-B irradiated Arabidopsis plants, we demonstrate that RPL10 associates with different proteins under the two different conditions, including nuclear proteins, suggesting that at least one isoform may have extra-ribosomal roles.

Ribosomal L10 Proteins in Plants

Ribosomal protein L10 (RPL10) participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome: it organizes the union site to aminoacyl-tRNA; but also its incorporation into the 60S subunit is required for the union of subunits and the initiation of translation.^1–4 However, increasing evidences demonstrate that RPL10 protein from various organisms has multiple extra-ribosomal functions, besides being a constituent of ribosome and its role in translation.^5–9

Depletion of stratospheric ozone has increased terrestrial UV-B levels with deleterious consequences for all living organisms, particularly for plants due to their sessile condition. UV-B photons generate photoproducts in DNA and can also directly damage proteins, lipids and RNA.^10–12 Thus, plants have evolved mechanisms of protection,¹³ repair,^14,15 and avoidance.^16,17 Although UV-B photon perception and signal transduction are largely unknown,^18–20 it was recently shown that the transcription factor long hypocotyl5 (HY5) is an important participant in the long wavelength (300 to 315 nm) UV-B-induced signal transduction cascade in Arabidopsis thaliana.²¹ Upregulation of HY5 is mediated by UVR8, in co-operation with COP1; UVR8 and COP1 interact directly and rapidly in the nucleus in planta after UV-B exposure.^22,23 It is proposed that this very early step in UV-B signaling coordinates responses, ensuring UV-B acclimation and protection.²⁴ In maize, by transcriptome profiling using different UV-B regimes, the functional group with the largest number of genes increased by UV-B corresponds to transcripts for proteins involved in translation, among them, rpl10, an homolog of the human QM transcript.²⁵ In addition, in maize and recently in A. thaliana, we demonstrated that UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B, restoring amino acid incorporation.^12,26

Regulation of Arabidopsis RPL10 Expression by UV-B Light

Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C that exhibit high similarity both at nucleotide and amino acid levels.²⁶ In addition, A. thaliana RPL10 proteins show high degree of primary sequence conservation with other eukaryotic orthologs; suggesting that RPL10 has been conserved during eukaryotic evolution. By qRT-PCR we have recently showed that, in maize and Arabidopsis plants, UV-B induced RPL10s expression, in agreement with our previous microarray results.^25,26 In order to study the UV-B regulation of AtRPL10 genes in detail, we did qRT-PCR experiments to analyze the effect of exposure under different UV-B fluences (0.5, 1 and 2 W m⁻²) for 4 h. Figure 1A shows that when plants are irradiated at 0.5 and 1 W m⁻², only RPL10C is upregulated by UV-B in mature leaves; however, when plants are treated with UV-B at a 2 W m⁻², RPL10C transcripts increases about six-fold, RPL10A expression is not changed; while RPL10B shows a decrease of 2.5-fold in its transcript levels. This result suggests that RPL10B expression is only repressed at higher UV-B intensities. The UV-B regulation of RPL10s expression was also studied at different times of UV-B exposure, from 1 to 4 h of UV-B at 2 W m⁻²; in addition, expression was investigated after a period of recovery in the absence of UV-B in plants that were UV-B irradiated for 4 h (Fig. 1B). Levels of RPL10C transcripts are increased with the time of exposure, for example after 3 h of UV-B, there is a more than two-fold increase in RPL10C transcripts than after 1 h of UV-B at the same intensity (Fig. 1B); similar results are obtained for RPL10B downregulation. 4 h after the end of the UV-B treatment, RPL10B transcript levels are similar to those in control plants that were not UV-B irradiated; although elevated RPL10C transcripts are still detected (2.5-fold higher than in control plants), but levels are 2.4-fold lower than those measured immediately after the UV-B treatment (Fig. 1B). It is worth mentioning that the evaluation of different stress parameters, like chlorophyll b, flavonoids or total proteins, both immediately after the UV-B treatments and after a period of recovery, indicated that UV-B radiation did not induce cell death at the wavelengths and fluence rates used in our experiments (not shown).

Figure 1.

Regulation of RPL10A–C expression by UV-B. (A) RPL10A–C expression after irradiation with different UV-B fluences (0.5, 1 and 2 W m⁻²) for 4 h in A. thaliana plants analyzed by qRT-PCR. (B) RPL10A–C expression after exposure under UV-B radiation for 1 to 4 h and after a period of recovery in the absence of UV-B (1–4 h recovery, 4 h UV-B treatment) analyzed by qRT-PCR. Each reaction was normalized using the C_t values corresponding to the CALCIUM PROTEIN KINASE 3 mRNA. The means of the results obtained using three independent RNA samples are shown, the error bars indicate the S.D. of the samples.

Co-immunoprecipitation of RPL10 Proteins

We recently showed by co-immunoprecipitation experiments followed by the identification of associated proteins by tandem mass spectrometry analysis that RPL10s in Arabidopsis associate with translation proteins, demonstrating it is a component of the 80S ribosome.²⁶ In addition, we have also identified groups of proteins that are involved in different metabolic processes in the cell, including nuclear proteins, suggesting that at least one of the RPL10 isoforms may have an extra-ribosomal function, for example associating with transcriptional activators or repressors as it was previously described in humans, E. histolytica and chicken.^8,27,28 Furthermore, we demonstrated that knockout rpl10A mutants are lethal, knockdown rpl10B mutants have abnormal growth and plant development and heterozygous rpl10A mutants are deficient in translation under UV-B radiation; clearly indicating that RPL10 genes are not functionally equivalent, and that they have important functions in development and UV-B responses. In order to explore putative RPL10 roles in UV-B responses, we conducted co-immunoprecipitation experiments using antibodies against human RPL10 and total protein extracts from Arabidopsis plants after 4 h of UV-B exposure and under control conditions in the absence of UV-B, and we compared associated proteins to RPL10 in both conditions. Proteins that were only associated to RPL10 either in control conditions in the absence of UV-B or after UV-B irradiation were selected and differential proteins were classified according to their functions (Fig. 2). Both under control conditions in the absence of UV-B, and after 4 h of UV-B, the functional group with the largest number of differential proteins is involved in translation, indicating that, as in our previous study, RPL10 is a constituent of the ribosome, and probably associates with different proteins in the ribosome under UV-B conditions (Fig. 2 and Sup. Table 1). In addition, we also identified differential proteins implicated in general processes of plant metabolism such as photosynthesis, respiration, amino acid, lipid, hormone and secondary metabolism, transport, signaling and stress response and detoxification, among others (Fig. 2 and Sup. Table 1). However, in the UV-B sample, we identified a higher percentage of proteins involved in DNA metabolism (duplication and chromatin structure) and RNA metabolism (transcription and regulation) suggesting that RPL10 may have a role in the nucleus under this stress condition. For example, under UV-B conditions, Arabidopsis RPL10 associates with histones, importin and exportin proteins involved in cytoplasm-nuclear trafficking and with transcriptional regulators like WRKY28 that participates in pathogen attack,²⁹ the basic transcriptional factor 3 (BTF3) involved in response to salt stress and geminivirus infection;^30,31 and the Tudor-2 protein with nuclease activity involved in the regulation of GA20Ox13 expression and the response to cadmium ion (Sup. Table 1).^32,33 Moreover, under UV-B conditions, RPL10 interacts with the GRP7 protein (glycine-rich RNA-binding protein), which has an important role in mediating innate immune response to pathogens,³¹ and the BTR1 protein that specifically binds to tomato mosaic virus (ToMV) genomic RNA and inhibits the local spread of ToMV.³⁴ Although ribosomes are assembled in the nucleolus, extensive research in yeast shows that RPL10 is loaded into the ribosome at a late step in maturation after the export of the 60S subunit to the cytoplasm.^3,4,35 Consequently, if the same situation occurs in plants, the presence of nuclear proteins co-immunoprecipitated with RPL10 would indicate its existence in the nucleus with an extra-ribosomal role. A. thaliana RPL10 proteins lack a nuclear localization signal; it is possible that these proteins can move to the nucleus associated with other proteins as the human QM protein.³⁶ In agreement with our hypothesis, it has been demonstrated that in A. thaliana plants, RPL10A protein localizes predominantly in the cytoplasm but also a small fraction resides in the nuclei.³⁷ RPL10A participates in plant defense responses against geminivirus, acting as a downstream effector of the transmembrane receptor NIK1. NIK1 phosphorylates RPL10A and relocates the cytosolic protein to the nucleus, negatively affecting geminivirus proliferation or movement. Thus, further studies of subcellular localization of GFP-fused RPL10s in Arabidopsis transgenic plants will allow demonstrating extraribosomal functions for these proteins in UV-B responses.

Figure 2.

Co-immunoprecipitation of RPL10 proteins in A. thaliana control plants (no UV-B) and after exposure under UV-B radiation for 4 h (UV-B). The graphs only show differential proteins. Co-immunoprecipitation experiments and LC-MS/MS were carried out as previously reported.²⁶ RPL10-associated proteins were classified based on their cell functions.

Addendum to: Falcone Ferreyra ML, Pezza A, Biarc J, Burlingame AL, Casati P. Plant L10 ribosomal proteins have different roles during development and translation under UV-B stress. Plant Physiol. 2010;153:1–17. doi: 10.1104/pp.110.157057.