Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.genome wide | high throughput | leaky scanning | start codon P rotein synthesis is the final step in the flow of genetic information and lies at the heart of cellular metabolism. Translation is regulated principally at the initiation stage, and during the last decade significant progress has been made in dissecting the role of initiation factors (eIFs) in the assembly of elongationcompetent 80S ribosomes (1-3). However, mechanisms underlying start codon recognition are not fully understood. Proper selection of the translation initiation site (TIS) on mRNAs is crucial for the production of desired protein products. A fundamental and longsought goal in understanding translational regulation is the precise determination of TIS codons across the entire transcriptome.In eukaryotes, ribosomal scanning is a well-accepted model for start codon selection (4). During cap-dependent translation initiation, the small ribosome subunit (40S) is recruited to the 5′ end of mRNA (the m 7 G cap) in the form of a 43S preinitiation complex (PIC). The PIC is thought to scan along the message in search of the start codon. It is commonly assumed that the first AUG codon that the scanning PIC encounters serves as the start site for translation. However, many factors influence the start codon selection. For instance, the initiator AUG triplet usually is in an optimal context, with a purine at position −3 and a guanine at position +4 (5). The presence of an mRNA secondary structure at or near the TIS position also influences the efficiency of recognition (6). In addition to these cis sequence elements, the stringency of TIS selection also is subject to regulation by trans-acting factors such as eIF1 and eIF1A (7,8). Inefficient recognition of an initiator codon results in a portion of 43S PIC continuing to ...

Cells have evolved exquisite mechanisms to fine-tune the rate of protein synthesis in response to stress. Systemic mapping of start codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we present quantitative translation initiation sequencing (QTI-seq), where the initiating ribosomes can be profiled in real time at single nucleotide resolution. The resultant initiation map not only delineates variations of start codon selection, but also highlights a dynamic range of initiation rates in response to nutrient starvation. The integrated data set provides unique insights into principles of alternative translation and mechanisms controlling different aspects of translation initiation. Using RiboTag mice, QTI-seq permits tissue-specific profiling of initiating ribosomes in vivo. Liver cell-specific ribosome profiling uncovers a robust translational reprogramming of the proteasome system in fasted mice. Our findings illuminate the prevalence and dynamic nature of translational regulation pivotal to physiological adaptation in vivo.

SUMMARY Translational control permits cells to respond swiftly to changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected mRNA fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and co-translational folding. Our results further support the chaperone “trapping” mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine-tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a co-translational stress response to maintain the intracellular protein homeostasis.

Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation factors, such as eIF4F and eIF2, and rapidly modulate mRNA translation at both the initiation and the elongation stages. Repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. The past decade has seen significant progress in our understanding of translational reprogramming in part due to the development of technologies that allow the dissection of the interplay between mRNA elements and corresponding binding proteins. Recent genome-wide studies using ribosome profiling have revealed unprecedented proteome complexity and flexibility through alternative translation, raising intriguing questions about stress-induced translational reprogramming. Many surprises emerged from these studies, including wide-spread alternative translation initiation, ribosome pausing during elongation, and reversible modification of mRNAs. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases.

Metal-organic frameworks (MOFs), which are typically embedded in polymer matrices as composites, are emerging as a new class of carriers for sustained drug delivery. Most of the MOFs and the polymers used so far in these composites, however, are not pharmaceutically acceptable. In the investigation reported herein, composites of γ-cyclodextrin (γ-CD)-based MOFs (CD-MOFs) and polyacrylic acid (PAA) were prepared by a solid in oil-in-oil (s/o/o) emulsifying solvent evaporation method. A modified hydrothermal protocol has been established which produces efficiently at 50 °C in 6 h micron (5-10 μm) and nanometer (500-700 nm) diameter CD-MOF particles of uniform size with smooth surfaces and powder X-ray diffraction patterns that are identical with those reported in the literature. Ibuprofen (IBU) and Lansoprazole (LPZ), both insoluble in water and lacking in stability, were entrapped with high drug loading in nanometer-sized CD-MOFs by co-crystallisation (that is more effective than impregnation) without causing MOF crystal degradation during the loading process. On account of the good dispersion of drug-loaded CD-MOF nanocrystals inside polyacrylic acid (PAA) matrices and the homogeneous distribution of the drug molecules within these crystals, the composite microspheres exhibit not only spherical shapes and sustained drug release over a prolonged period of time, but they also demonstrate reduced cell toxicity. The cumulative release rate for IBU (and LPZ) follows the trend: IBU-γ-CD complex microspheres (ca. 80% in 2 h) > IBU microspheres > IBU-CD-MOF/PAA composite microspheres (ca. 50% in 24 h). Importantly, no burst release of IBU (and LPZ) was observed from the CD-MOF/PAA composite microspheres, suggesting an even distribution of the drug as well as strong drug carrier interactions inside the CD-MOF. In summary, these composite microspheres, composed of CD-MOF nanocrystals embedded in a biocompatible polymer (PAA) matrix, constitute an efficient and pharmaceutically acceptable MOF-based carrier for sustained drug release.

No abstract

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA binding protein whose deficiency impacts many brain functions, including differentiation of adult neural stem cells (aNSCs). However, the mechanism by which FMRP influences these processes remains unclear. Here, we performed ribosome profiling and transcriptomic analysis of aNSCs in parallel from wild-type and Fmr1 knockout mice. Our data revealed diverse gene expression changes at both mRNA and translation levels. Many mitosis and neurogenesis genes were dysregulated primarily at the mRNA level, while numerous synaptic genes were mostly dysregulated at the translation level. Translational “buffering”, whereby changes in ribosome association with mRNA are compensated by alterations in RNA abundance, was also evident. Knockdown of NECDIN, an FMRP-repressed transcriptional factor, rescued neuronal differentiation. In addition, we discovered that FMRP regulates mitochondrial mRNA expression and energy homeostasis. Thus, FMRP controls diverse transcriptional and posttranscriptional gene expression programs critical for neural differentiation.

Microwave-assisted rapid synthesis of γ-cyclodextrin metal-organic 2 frameworks for size control and efficient drug loading 3 4