In prokaryotes and eukaryotes, most genes appear to be transcribed during short periods called transcriptional bursts, interspersed by silent intervals. We describe how such bursts generate gene-specific temporal patterns of messenger RNA (mRNA) synthesis in mammalian cells. To monitor transcription at high temporal resolution, we established various gene trap cell lines and transgenic cell lines expressing a short-lived luciferase protein from an unstable mRNA, and recorded bioluminescence in real time in single cells. Mathematical modeling identified gene-specific on- and off-switching rates in transcriptional activity and mean numbers of mRNAs produced during the bursts. Transcriptional kinetics were markedly altered by cis-regulatory DNA elements. Our analysis demonstrated that bursting kinetics are highly gene-specific, reflecting refractory periods during which genes stay inactive for a certain time before switching on again.

Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.

In mammalian tissues, circadian gene expression can be driven by local oscillators or systemic signals controlled by the master pacemaker in the suprachiasmatic nucleus. We show that simulated body temperature cycles, but not peripheral oscillators, controlled the rhythmic expression of cold-inducible RNA-binding protein (CIRP) in cultured fibroblasts. In turn, loss-of-function experiments indicated that CIRP was required for high-amplitude circadian gene expression. The transcriptome-wide identification of CIRP-bound RNAs by a biotin-streptavidin-based cross-linking and immunoprecipitation (CLIP) procedure revealed several transcripts encoding circadian oscillator proteins, including CLOCK. Moreover, CLOCK accumulation was strongly reduced in CIRP-depleted fibroblasts. Because ectopic expression of CLOCK improved circadian gene expression in these cells, we surmise that CIRP confers robustness to circadian oscillators through regulation of CLOCK expression.

SummaryProtein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington’s disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

Defi ciency in plant-available phosphorus is considered to be a major limiting factor to food production in many agricultural soils. Mineral resources are necessary to restore soil phosphorus content. In regions where conventional fertilizers are not used due to cost limitations or to mitigate adverse environmental effects, local sources of phosphate rock are being increasingly recognized for potential use as alternative phosphorus fertilizers. The main obstacle associated with using directly applied ground phosphate rock is that the phosphate released is often unable to supply suffi cient plant-available phosphorus for crop uptake. Plantand microbial-based mechanisms are low-cost, appropriate technologies to enhance the solubilization and increase the agronomic effectiveness of phosphate rock. Common mechanisms of phosphate rock dissolution including proton and organic acid production will be reviewed for both plants and microorganisms. This review will also address possibilities for future research directions and applications to agriculture, as well as highlight ongoing research at the University of Guelph, Guelph, Canada.

Cells and organisms have evolved numerous mechanisms to cope with and to adapt to unexpected challenges and harsh conditions. Proteins are essential to perform the vast majority of cellular and organismal functions. To maintain a healthy proteome, cells rely on a network of factors and pathways collectively known as protein quality control (PQC) systems, which not only ensure that newly synthesized proteins reach a functional conformation but also are essential for surveillance, prevention, and rescue of protein defects. The main players of PQC systems are chaperones and protein degradation systems: the ubiquitin-proteasome system and autophagy. Here we provide an integrated overview of the diverse PQC systems in eukaryotic cells in health and diseases, with an emphasis on the key regulatory aspects and their cross talks. We also highlight how PQC regulation may be exploited for potential therapeutic benefit.

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Organisms have evolved mechanisms to cope with and adapt to unexpected challenges and harsh conditions. Unfolded or misfolded proteins represent a threat for cells and organisms, and the deposition of misfolded proteins is a defining feature of many age-related human diseases, including the increasingly prevalent neurodegenerative diseases. These protein misfolding diseases are devastating and currently cannot be cured, but are hopefully not incurable. In fact, the aggregation-prone and potentially harmful proteins at the origins of protein misfolding diseases are expressed throughout life, whereas the diseases are late onset. This reveals that cells and organisms are normally resilient to disease-causing proteins and survive the threat of misfolded proteins up to a point. This Commentary will outline the limits of the cellular resilience to protein misfolding, and discuss the possibility of pushing these limits to help cells and organisms to survive the threat of misfolding proteins and to avoid protein quality control catastrophes.