A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

PPARγ coactivator-1α contributes to exercise-induced regulation of intramuscular lipid droplet programming in mice and humans.

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.

Rapid commitment to negative selection of a thymocyte line independent of CD4 co-engagement.

Biochemical and genetic investigations of thymocyte negative selection will be facilitated by the availability of cultured cell lines representing immature thymocytes, but to date thymocyte lines have demonstrated resistance to Ag receptor-mediated death. It is shown here that a CD4+CD8+ TCR alpha beta+ cell line with a TCR of known Ag and class II MHC specificity (cytochrome c with I-Ek) undergoes Ag dose-dependent apoptotic death in a manner consistent with negative selection of normal thymocytes. In contrast to superantigen-induced selection, apoptosis in this model is unaffected by blocking co-engagement of CD4 and the TCR. At high Ag doses, a significant fraction of thymocytes becomes committed to apoptosis within 2 h of contact with antigen-pulsed APCs, while increased interaction times increase the probability of death. Longer contact periods are required at lower Ag doses. These kinetics of commitment to apoptosis suggest that, in normal negative selection, a thymocyte clone may be deleted after any of several encounters with MHC-bearing cells during progress through the thymus. They also suggest why negative selection predominates when a specific peptide is competent both to delete and to select positively.

The alloreactive and self-restricted CD4+ T cell response directed against a single MHC class II/peptide combination.

The cellular basis for allograft rejection derives from the strong T cell response to cells bearing foreign MHC. While it was originally assumed that alloreactive T cells focus their recognition on the polymorphic residues that differ between syngeneic and allogeneic MHC molecules, studies with MHC class I-restricted CTL have shown that MHC-bound peptides play a critical role in allorecognition. It has been suggested that alloreactive T cells depend more strongly on interactions with the MHC molecule than with the associated peptide, but there is little evidence to support this idea. Here we have studied the alloreactive and self-restricted response directed against the class II H2-Ab molecule bound with a single peptide, Ep, derived from the H2-Ealpha chain. This MHC class II-peptide combination was a poor target and stimulator of alloreactive CD4+ T cell responses, indicating that MHC-bound peptides are as important for alloreactive CD4+ T cells as they are for alloreactive CTL. We also generated alloreactive T cells with exquisite specificity for the Ab/Ep complex, and compared their reactivity with self-restricted T cells specific for the same Ab/Ep complex. Our results showed that peptide-specific alloreactive T cells, as compared with self-restricted T cells, were more sensitive to peptide stimulation, but equally sensitive to amino acid substitutions in the peptide. These findings indicate that alloreactive and self-restricted T cells interact similarly with their MHC/peptide ligand.

Peptide dependency of alloreactive CD4+ T cell responses.

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.