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Phosphorylation of ribosomal protein S6 differentially affects mRNA translation based on ORF length.

ABSTRACT: Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5' TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.

SUBMITTER: Bohlen J

PROVIDER: S-EPMC8682771 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

Project description:Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, its molecular consequences on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. Loss of RPS6 phosphorylation causes a correspondingly larger drop in translation efficiency of mRNAs with short CDSs than long CDSs. Interestingly, mRNAs with 5’ TOP motifs are translated well also in the absence of RPS6 phosphorylation despite short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.

2021-12-22 | GSE168977 | GEO

Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

Project description:Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

| PRJNA714824 | ENA

Ribosomal Protein S6 Phosphorylation Is Involved in Novelty-Induced Locomotion, Synaptic Plasticity and mRNA Translation.

Project description:The phosphorylation of the ribosomal protein S6 (rpS6) is widely used to track neuronal activity. Although it is generally assumed that rpS6 phosphorylation has a stimulatory effect on global protein synthesis in neurons, its exact biological function remains unknown. By using a phospho-deficient rpS6 knockin mouse model, we directly tested the role of phospho-rpS6 in mRNA translation, plasticity and behavior. The analysis of multiple brain areas shows for the first time that, in neurons, phospho-rpS6 is dispensable for overall protein synthesis. Instead, we found that phospho-rpS6 controls the translation of a subset of mRNAs in a specific brain region, the nucleus accumbens (Acb), but not in the dorsal striatum. We further show that rpS6 phospho-mutant mice display altered long-term potentiation (LTP) in the Acb and enhanced novelty-induced locomotion. Collectively, our findings suggest a previously unappreciated role of phospho-rpS6 in the physiology of the Acb, through the translation of a selective subclass of mRNAs, rather than the regulation of general protein synthesis.

| S-EPMC5742586 | biostudies-literature

The putative RNA helicase HELZ promotes cell proliferation, translation initiation and ribosomal protein S6 phosphorylation.

Project description:The hypoxia-inducible transcription factor (HIF) is a key component of the cellular adaptation mechanisms to hypoxic conditions. HIF? subunits are degraded by prolyl-4-hydroxylase domain (PHD) enzyme-dependent prolyl-4-hydroxylation of LxxLAP motifs that confer oxygen-dependent proteolytic degradation. Interestingly, only three non-HIF? proteins contain two conserved LxxLAP motifs, including the putative RNA helicase with a zinc finger domain HELZ. However, HELZ proteolytic regulation was found to be oxygen-independent, supporting the notion that a LxxLAP sequence motif alone is not sufficient for oxygen-dependent protein destruction. Since biochemical pathways involving RNA often require RNA helicases to modulate RNA structure and activity, we used luciferase reporter gene constructs and metabolic labeling to demonstrate that HELZ overexpression activates global protein translation whereas RNA-interference mediated HELZ suppression had the opposite effect. Although HELZ interacted with the poly(A)-binding protein (PABP) via its PAM2 motif, PABP was dispensable for HELZ function in protein translation. Importantly, downregulation of HELZ reduced translational initiation, resulting in the disassembly of polysomes, in a reduction of cell proliferation and hypophosphorylation of ribosomal protein S6.

| S-EPMC3135610 | biostudies-literature

Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor progression in distal cholangiocarcinoma.

Project description:BackgroundThe prognosis of distal cholangiocarcinoma (dCCA) remains poor; thus, the identification of new therapeutic targets is warranted. Phosphorylated S6 ribosomal protein indicates a mammalian target of rapamycin complex 1 (mTORC1) activity, and mTORC1 plays a central role in controlling cell growth and regulating glucose metabolism. We aimed to clarify the effect of S6 phosphorylation on tumor progression and the glucose metabolic pathway in dCCA.MethodsThirty-nine patients with dCCA who underwent curative resection were enrolled in this study. S6 phosphorylation and the expression of GLUT1 were evaluated by immunohistochemistry, and their relationship with clinical factors was investigated. The effect of S6 phosphorylation on glucose metabolism with PF-04691502 treatment, an inhibitor of S6 phosphorylation, was examined in cancer cell lines by Western blotting and metabolomics analysis. Cell proliferation assays were performed with PF-04691502.ResultsS6 phosphorylation and the expression of GLUT1 were significantly higher in patients with an advanced pathological stage. Significant correlations between GLUT1 expression, S6 phosphorylation, and SUV-max of FDG-PET were shown. In addition, cell lines with high S6 phosphorylation levels showed high GLUT1 levels, and the inhibition of S6 phosphorylation reduced the expression of GLUT1 on Western blotting. Metabolic analysis revealed that inhibition of S6 phosphorylation suppressed pathways of glycolysis and the TCA cycle in cell lines, and then, cell proliferation was effectively reduced by PF-04691502.ConclusionUpregulation of glucose metabolism via phosphorylation of S6 ribosomal protein appeared to play a role in tumor progression in dCCA. mTORC1 may be a therapeutic target for dCCA.

| S-EPMC10190040 | biostudies-literature

Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

Project description:The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

| S-EPMC4344886 | biostudies-literature

Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.

Project description:In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the integrated stress response (ISR). The ISR increases the expression of genes important for alleviating stress or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this study, we show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5'-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF due to its poor start site context, and instead it allows scanning ribosomes to translate CHOP. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.

| S-EPMC3064149 | biostudies-literature

Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2).

Project description:Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2-which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome-can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK.

| S-EPMC5061434 | biostudies-literature

Stimulation of skeletal muscle myofibrillar protein synthesis, p70 S6 kinase phosphorylation, and ribosomal protein S6 phosphorylation by inhibition of myostatin in mature mice.

Project description:Knocking out myostatin activity during development increases the rate of muscle protein synthesis. The present study was done to determine whether postdevelopmental loss of myostatin activity stimulates myofibrillar protein synthesis and the phosphorylation of some of the proteins involved in regulation of protein synthesis rate. Myostatin activity was inhibited for 4 days, in 4- to 5-mo-old male mice, with injections of an anti-myostatin antibody (JA16). The mean myofibrillar synthesis rate increased 19% (P < 0.01) relative to the mean rate in saline-treated mice, as determined by incorporation of deuterium-labeled phenylalanine. JA16 increased phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6 (rpS6) 1.9-fold (P < 0.05). It did not affect phosphorylation of eukaryotic initiation factor 4E-binding protein-1 or Akt. Microarrays and real-time PCR analyses indicated that JA16 administration did not selectively enrich levels of mRNAs encoding myofibrillar proteins, ribosomal proteins, or translation initiation and elongation factors. Rapamycin treatment did not affect the rate of myofibrillar protein synthesis whether or not the mice received JA16 injections, although it eliminated the phosphorylation of S6K and rpS6. We conclude that the normal level of myostatin activity in mature muscle is sufficient to inhibit myofibrillar synthesis rate and phosphorylation of S6K and rpS6. Reversal of the inhibition of myofibrillar synthesis with an anti-myostatin antibody is not dependent on mTOR activation.

| S-EPMC2660146 | biostudies-literature

Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation.

Project description:Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.

| S-EPMC4795744 | biostudies-literature

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