Summary Sickle cell disease (SCD), caused by a mutation in the β-globin gene HBB, is widely distributed in malaria endemic regions. Cardiopulmonary complications are major causes of morbidity and mortality. Hemoglobin SS (Hb SS) represents a large proportion of SCD in the Americas, United Kingdom, and certain regions of Africa while higher proportions of hemoglobin SC are observed in Burkina Faso and hemoglobin Sβ-thalassemia in Greece and India. Coinheritance of α-thalassemia and persistence of hemoglobin F production are observed in highest frequency in certain regions of India and the Middle East. As confirmed in the PUSH and Walk-PHaSST studies, Hb SS, absence of co-inheriting alpha-thalassemia, and low hemoglobin F levels tend to be associated with more hemolysis, lower hemoglobin oxygen saturations, greater proportions of elevated tricuspid regurgitant jet velocity and brain natriuretic peptide, and increased left ventricular mass index. Identification of additional genetic modifiers will improve prediction of cardiopulmonary complications in SCD.
An early signaling event activated by amino acids and growth factors in many cell types is the phosphorylation of the mammalian target of rapamycin (mTOR; FRAP), which is functionally linked to ribosomal protein s6 kinase (p70 s6k ), a kinase that plays a critical regulatory role in the translation of mRNAs and protein synthesis. We previously showed that intestinal cell migration, the initial event in epithelial restitution, is enhanced by L-arginine (ARG). In this study, we used amino acids as prototypic activators of mTOR and ARG, IGF-1, or serum as recognized stimulators of intestinal cell migration. We found that 1) protein synthesis is required for intestinal cell migration, 2) mTOR/ p70 s6k pathway inhibitors (rapamycin, wortmannin, and intracellular Ca 2 + chelation) inhibit cell migration, 3) ARG activates migration and mTOR/p70 s6k (but not ERK-2) in migrating enterocytes, and 4) immunocytochemistry reveals abundant p70 s6k staining in cytoplasm, whereas phosphop70 s6k is virtually all intranuclear in resting cells but redistributes to the periphery on activation by ARG. We conclude that mTOR/p70 s6k signaling is essential to intestinal cell migration, is activated by ARG, involves both nuclear and cytoplasmic events, and may play a role in intestinal repair. Keywordsinsulin-like growth factor-1; arginine; p70 s6 kinase; extracellular signal-regulated kinase-2; rapamycin Acute regulation of protein synthesis in mammalian cells is achieved through changes in the rate of translation of mRNA via alterations in peptide chain initiation. A critical step in this process is binding of mRNA to the 43S preinitiation complex. Two key translational regulators in mammalian cells are ribosomal protein s6 kinase (p70 s6k ) and 4E-BP1, both of which are upstream of preinitiation. Both p70 s6k and 4E-BP1 are tightly regulated by the mammalian target of rapamycin (mTOR). mTOR has been called the "nutrient sensor" of cells, capable of integrating environmental factors with an organism's ability to survive (8,20). Peterson et al. (25) reported that amino acids stimulate an inhibitory interaction between mTOR and protein phosphatase 2A, placing p70 s6k in the activated state. Conversely, mTOR is inhibited by individual and global amino acid withdrawal. Rapamycin (at nanomolar concentrations) blocks p70 s6k activation by amino acids (15) and other agonists.Address for reprint requests and other correspondence: M. Rhoads, Div.
Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs indcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed thatdcp2Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased bydcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs indcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated bydcp2Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.
We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1Δ and pat1Δ mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in wild-type cells. These mRNAs show both reduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1. mRNA degradation stimulated by Dhh1/Pat1 is not dictated by poor translation nor enrichment for suboptimal codons. Pat1 and Dhh1 also collaborate to reduce translation and protein production from many mRNAs. Transcripts showing concerted translational repression by Pat1/Dhh1 include mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress numerous transcripts involved in respiration, catabolism of non-preferred carbon or nitrogen sources, or autophagy; and we obtained evidence for elevated respiration and autophagy in the mutants. Thus, Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.
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