Effective T cell responses against invading pathogens require the concerted production of three key cytokines: TNF-α, IFN-γ, and IL-2. The cytokines functionally synergize, but their production kinetics widely differ. How the differential timing of expression is regulated remains, however, poorly understood. We compared the relative contribution of transcription, mRNA stability, and translation efficiency on cytokine production in murine effector and memory CD8 + T cells. We show that the immediate and ample production of TNF-α is primarily mediated by translation of preformed mRNA through protein kinase C (PKC)-induced recruitment of mRNA to polyribosomes. Also, the initial production of IFN-γ uses translation of preformed mRNA. However, the magnitude and subsequent expression of IFN-γ, and of IL-2, depends on calcium-induced de novo transcription and PKC-dependent mRNA stabilization. In conclusion, PKC signaling modulates translation efficiency and mRNA stability in a transcript-specific manner. These cytokine-specific regulatory mechanisms guarantee that T cells produce ample amounts of cytokines shortly upon activation and for a limited time.T cells | cytokine production | PKC | mRNA stability | translation efficiency

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.

Expression of the RNA-binding protein Csde1 (Cold shock domain protein e1) is strongly upregulated during erythropoiesis compared to other hematopoietic lineages. Csde1 expression is impaired in the severe congenital anemia Diamond Blackfan Anemia (DBA), and reduced expression of Csde1 in healthy erythroblasts impaired their proliferation and differentiation. To investigate the cellular pathways controlled by Csde1 in erythropoiesis, we identified the transcripts that physically associate with Csde1 in erythroid cells. These mainly encoded proteins involved in ribogenesis, mRNA translation and protein degradation, but also proteins associated with the mitochondrial respiratory chain and mitosis. Crispr/ Cas9-mediated deletion of the first cold shock domain of Csde1 affected RNA expression and/or protein expression of Csde1-bound transcripts. For instance, protein expression of Pabpc1 was enhanced while Pabpc1 mRNA expression was reduced indicating more efficient translation of Pabpc1 followed by negative feedback on mRNA stability. Overall, the effect of reduced Csde1 function on mRNA stability and translation of Csde1-bound transcripts was modest. Clones with complete loss of Csde1, however, could not be generated. We suggest that Csde1 is involved in feed-back control in protein homeostasis and that it dampens stochastic changes in mRNA expression.RNA binding proteins (RBP) regulate transcript stability and translation. RBPs can cooperate with protein complexes of the general mRNA translation machinery, or with protein complexes that control mRNA location and/ or degradation. Deregulated expression of such RBPs affects protein synthesis from a set of transcripts particularly dependent on that specific RBP. This has been referred to as an RNA regulon 1 . The RNA regulon may be cell-type specific, because it depends on the available transcriptome in these cells. The RNA regulon may also define a set of ubiquitously expressed transcripts whose translation is modified by cell type specific expression of RBP. For instance, hematopoietic stem-and progenitor cells (HSPC) express the RBP Musashi-2, which is crucial for maintenance and repopulation ability of the HSPC 2 . The RBP Znf36l2 (Zinc finger protein 36-like 2), a member of the Tristetraproline family, is expressed in early erythroblasts and mediates glucocorticoid-mediated expansion of the erythroid compartment 3 . We observed that the RBP Csde1 is strongly upregulated in expanding erythroblasts (>100-fold) compared to other hematopoietic cell types 4 . In Diamond Blackfan Anemia (DBA), a ribosomopathy due to haploinsufficiency of ribosomal proteins, this upregulation was impaired 4 . This raised our interest in the role of Csde1 in erythropoiesis.Csde1 was first described as Unr (upstream of N-ras) in Drosophila melanogaster 5 . It binds an AG-rich domain in the 3′UTR of Msl (Male sex lethal) and suppresses translation 6,7 . Its expression is highly conserved across species and Csde1 binds its own IRES to repress translation in mammalian cells 8 . Csde1 also ...

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.

Diamond–Blackfan anaemia (DBA) is a congenital disorder that presents in the first year of life as severe anaemia, and in several patients is coupled with developmental defects. DBA is a ribosomopathy because almost all known mutations or deletions occur in genes encoding ribosomal proteins (RPs) that impair ribosome biogenesis. However, atypical examples of patients carry mutations in the erythroid specific transcription factor GATA1. DBA is a rare disease that displays a high level of heterogeneity with respect to the affected RP and disease penetrance. The reduced availability of ribosomes affects several cellular processes, including stabilisation of the p53 tumour suppressor, and impaired messenger ribonucleic acid (mRNA) translation. Registration of the genetic and phenotypic characteristics of DBA patients worldwide is needed to understand the relation between mutations, patient symptoms and cellular processes that underlie this pathophysiology. Key Concepts Diamond–Blackfan anaemia (DBA) is caused by haploinsufficiency of one of several ribosomal proteins in at least two‐thirds of all patients; the disease causing mutation is unknown in approximately 30% of patients. The mechanisms underlying the ribosomopathy DBA are multiple, and are incompletely understood. Particularly, the roles of transcript specific translation and iron homeostasis are underestimated. Defective mRNA translation in ribosomopathies such as DBA should be investigated in cells that are affected in the disease, because an mRNA translation defect depends on the cell's specific transcriptome. DBA type I and DBA type II should be used to distinguish between patients carrying RP mutations versus those carrying mutations in genes that specifically affect erythropoiesis including GATA1 . The ribosomopathy Diamond–Blackfan anaemia should be renamed to Diamond–Blackfan syndrome to emphasise that it is a systemic disease of which severe anaemia at young age is the most common, but not the exclusive symptom. The heterogeneity with which DBA presents, even when family members carry the same mutation, needs to be investigated to find a proper treatment for severe DBA.

Expression of the RNA-binding protein Csde1 (Cold shock domain protein e1) is strongly upregulated during erythropoiesis compared to other hematopoietic lineages. In the severe congenital anemia Diamond Blackfan Anemia (DBA), however, Csde1 expression is impaired. Reduced expression of Csde1 in healthy erythroblasts impaired their proliferation and differentiation, which suggests an important role for Csde1 in erythropoiesis. To investigate the cellular pathways controlled by Csde1 in erythropoiesis, we identified the transcripts that physically associate with Csde1 in erythroid cells. These mainly encoded proteins involved in ribogenesis, mRNA translation and protein degradation, but also proteins associated with the mitochondrial respiratory chain and mitosis. Crispr/Cas9-mediated deletion of the first cold shock domain of Csde1 affected RNA expression and/or protein expression of Csde1-bound transcripts. For instance, protein expression of Pabpc1 was enhanced while Pabpc1 mRNA expression was reduced indicating more efficient translation of Pabpc1 followed by negative feedback on mRNA stability. Overall, the effect of reduced Csde1 function on mRNA stability and translation of Csde1-bound transcripts was modest. Clones with complete loss of Csde1, however, could not be generated. We suggest that Csde1 is involved in feed-back control in protein homeostasis and that it dampens stochastic changes in mRNA expression.

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how eIF2 phosphorylation affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict eIF2-dependent translation efficiency, but uORF identity differs. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.- eif2 dependent translation in erythroblasts during proteotoxic stress determined by ribosome footprinting- identification of transcription factors upregulated in response to eIF2 phosphorylation- Advantages and disadvantages of translation initiation site determination using harringtonine- distinct uORF pattern in transcripts with enhanced, or more than average reduced translation upon proteotoxic stress