Involvement of Target of Rapamycin (TOR) Signaling in the Regulation of Crosstalk between Ribosomal Protein Small Subunit 6 Kinase-1 (RPS6K-1) and Ribosomal Proteins

Abstract

The target of rapamycin (TOR) protein phosphorylates its downstream effector p70kDa ribosomal protein S6 kinases (S6K1) for ribosome biogenesis and translation initiation in eukaryotes. However, the molecular mechanism of TOR-S6K1-ribosomal protein (RP) signaling is not well understood in plants. In the present study, we report the transcriptional upregulation of ribosomal protein large and small subunit (RPL and RPS) genes in the previously established TOR overexpressing transgenic lines of rice (in Oryza sativa ssp. indica, variety BPT-5204, TR-2.24 and TR-15.1) and of Arabidopsis thaliana (in Col 0 ecotype, ATR-1.4.27 and ATR-3.7.32). The mRNA levels of RP genes from this study were compared with those previously available in transcriptomic datasets on the expression of RPs in relation to TOR inhibitor and in the TOR-RNAi lines of Arabidopsis thaliana. We further analyzed TOR activity, i.e., S6K1 phosphorylation in SALK lines of Arabidopsis with mutation in rpl6, rpl18, rpl23, rpl24 and rps28C, where the rpl18 mutant showed inactivation of S6K1 phosphorylation. We also predicted similar putative Ser/Thr phosphorylation sites for ribosomal S6 kinases (RSKs) in the RPs of Oryza sativa ssp. indica and Arabidopsis thaliana. The findings of this study indicate that the TOR pathway is possibly interlinked in a cyclic manner via the phosphorylation of S6K1 as a modulatory step for the regulation of RP function to switch 'on'/'off' the translational regulation for balanced plant growth.

Keywords: ribosomal proteins large subunit genes; ribosomal proteins small subunit genes; target of rapamycin.

Figure 1

Heatmap representation of expression of RP genes in the TOR-OE lines. Heatmap represents hierarchical clustering of the expression of RPL and RPS genes in TOR-overexpressing lines of rice and Arabidopsis. The RT-qPCR is used to determine the expression levels of (a) RPL and RPS genes in the TOR-OE lines TR-2.24 and TR-15.1 of rice and (b) in the lines ATR-1.4.27 and ATR-3.7.32 of Arabidopsis. Red and orange colors indicate increased RP gene expression, and the yellow color indicates decreased RP gene expression. The fold change was normalized using the 2^−∆∆CT method relative to their corresponding WT control.

Figure 2

Transcriptional regulation of ribosomal protein large subunit (RPL) and small subunit (RPS) genes in the TOR-OE lines of rice and Arabidopsis. The 7 DAG plants of two high AtTOR-expressing rice transgenic lines TR-2.24 and TR-15.1 were used to analyze the expression of RPL and RPS genes. The WT plants were used as controls. (a) Expression analysis of rice RPL genes in two high AtTOR overexpression lines of rice, TR-2.24 and TR-15.1. The RPL4, RPL14, RPL18A, RPL19.3, RPL36.2 and RPL51 genes were highly upregulated by 20-fold in both of the transgenic lines. (b) Expression analysis of rice RPS genes in two transgenic lines. The significant upregulation of RPS gene transcripts in two transgenic lines was observed, where the RPS3A, RPS6, RPS6A, RPS25A and RPS30 genes were highly upregulated by more than 7-fold in the transgenic plants. (c,d) The expression of RPL and RPS genes was also analyzed in two TOR-OE transgenic lines, ATR-1.4.27 and ATR-3.7.32, of Arabidopsis. The fold change was normalized using the ΔΔCT method relative to the WT plants. Rice Actin (Act1) and Arabidopsis Actin (Act2) were used as internal controls. Three biological and three technical replicates were included in this study. Vertical bars indicate the mean ± SE of three independent experiments and ANOVA analysis indicated the statistically significant differences, represented by asterisks (*) p < 0.05 and (**) p < 0.001.

Figure 3

Comparison of the overlapping expression patterns of RPL and RPS genes in TOR-OE lines of rice and Arabidopsis with the AZD-8055-treated Arabidopsis lines. The RP genes exhibiting a transcript level of ≥1-fold on log₂ scale were considered as significantly upregulated and the transcript level below 1-fold was considered downregulated or unchanged in expression. Venn diagrams are used to show the overlaps between the RP gene expression (a) in the AZD-8055-treated lines of Arabidopsis (Dong et al., 2015 [27]) and TOR-OE lines of rice (TR-2.24 and TR-15.1) and (b) in the AZD-8055-treated lines of Arabidopsis and TOR-OE Arabidopsis lines, ATR-1.4.27 and ATR-3.7.32.

Figure 4

Comparison of the overlapping expression patterns of RPL and RPS genes in TOR-OE lines of rice and Arabidopsis with the AtTOR-RNAi Arabidopsis lines. The RP genes exhibiting a transcript level of ≥1-fold on log₂ scale were considered as significantly upregulated and the transcript level below 1-fold was considered downregulated or unchanged in expression. Venn diagrams are used to show the overlaps between the RP gene expression (a) in the TOR-RNAi lines of Arabidopsis (Dobrenel et al., 2016 [19]) and TOR-OE lines of rice, TR-2.24 and TR-15.1, (b) in the TOR-RNAi lines of Arabidopsis and TOR-OE Arabidopsis lines, ATR-1.4.27 and ATR-3.7.32, and (c) in the TOR-OE lines of rice, TR-2.24 and TR-15.1, and ATR-1.4.27 and ATR-3.7.32 Arabidopsis transgenic lines.

Figure 5

S6K1 phosphorylation assay in Arabidopsis T- DNA insertional mutants. Phosphorylation of p70kDa S6K1 in Thr389 residue was detected in Arabidopsis T-DNA insertional mutants of tor and s6k1 protein along with mutants of ribosomal proteins rpl6, rpl18, rpl23a, rpl24 and rps28, and total protein isolated from WT (Col 0) Arabidopsis was taken as the control. (a) Phospho S6K1 detection in all of the mutants; (b) Western blot analysis of total S6K protein; (c) GAPDH protein used as loading control; (d) ponceau stain of the blot (e) the relative band intensity of S6K1 phosphorylation and (f) total S6K1 to band intensity of GAPDH was analyzed in the Arabidopsis T-DNA insertion mutants using Image J software. One-way ANOVA analysis was performed using the mean values (n = 3) with ±SE. Significantly reduced S6K1 phosphorylation compared with Col0 (WT, control) at p < 0.001 are marked with asterisks (*).

Figure 6

Possible feedback regulation of S6K1 phosphorylation via the TOR pathway. An illustration of TOR Complex 1-mediated regulation of S6K1 phosphorylation and translational initiation by further phosphorylation and activation of RPS6 protein and other RPL and RPS proteins. Dashed lines represent signaling pathways or intermediates that are not fully revealed. The observations from the predicted PPI networks and the inhibition of RP genes suggest that the S6K1 phosphorylation is differentially regulated. Possibly, the TOR and RPs are interlinked for the regulation of S6K1 phosphorylation, where RPs also have an independent role in differentially regulating the S6K phosphorylation and modulating protein translation in the plant cell. The figure represents a model for the regulation of S6K1 phosphorylation by loss of RPs in plants, which is possibly mediated via the association of RPs with the S6K1 protein or the other regulatory proteins in the TOR pathway. The illustration was created using www.biorender.com (accessed on 21 December 2022) and exported using a free trial subscription.