SUMMARY The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells with the site of delivey determined solely by a co-expressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence and reveal the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Summary The conserved transcriptional regulator Heat Shock Factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol, yet its regulation is poorly understood. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC and Hsf1 senses an RQC-mediated translation stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.

Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system. Studies in many biological systems have identified positive feedback as the key regulatory motif in the creation of switches with all-or-none "digital" output characteristics (1). Although a single positive feedback loop (A activates B and B activates A) or the equivalent double-negative feedback loop (A inhibits B and B inhibits A) can, under the proper circumstances, generate a bistable all-or-none switch (1-5), it is intriguing that many biological systems have not only a single but multiple positive feedback loops (Table 1). Three examples of positive feedback systems are shown in more detail in Fig. 1. Polarization in budding yeast depends on two positive feedback loops, a rapid loop involving activity cycling of the small guanosine triphosphatase Cdc42 and a slower loop that may involve actin-mediated transport of Cdc42 (Fig. 1A) (6). In many cell types, the induction of prolonged Ca 2+ signals involves initial rapid positive feedback loops centered on Ca 2+ release mediated by inositol 1,4,5-trisphosphate (IP3) combined with a much slower loop that induces Ca 2+ influx mediated by the depletion of Ca 2+ stores (7,8) (Fig. 1B). Xenopus oocytes respond to maturation-inducing stimuli by activating a rapid phosphorylation/dephosphorylation-mediated positive feedback loop (between Cdc2, Myt1, and Cdc25) and a slower translational positive feedback loop [between Cdc2 and the the mitogen-activated protein kinase (MAPK or ERK) cascade, which includes Mos, MEK (MAPK kinase), and p42] (Fig. 1C).The presence of multiple interlinked positive loops raises the question of the performance advantage of the multiple-loop design. One clue is provided by recent studies of budding yeast polarization. When the slow positive feedback loop is selectively compromised by treatment with the actin-depolymerizing agent latrunculin, the result is rapid but unstable NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cell polarization (6). In contrast, cells lacking a functional fast loop (by deletion of Bem1) form stable poles, but with reduced speed (6). These experimental observations led us to hypothesize that the slow positive feedback loop is crucial for the stability of the polarized "on" state, whereas the fast loop is critical for the speed of the transition between the unpolarized "off" state and polarized on state.To test this hypothesis computationally, we created models of positive feedback switches containing ...

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”).

Positive and negative feedback loops are common regulatory elements in biological signaling systems. We discuss core feedback motifs that have distinct roles in shaping signaling responses in space and time. We also discuss approaches to experimentally investigate feedback loops in signaling systems.Feedback loops are processes that connect output signals back to their inputs. The history of biological feedback goes back at least 130 years to observations by Eduard Pflüger that organs and other living systems "satisfy their own needs" (1). Feedback became an influential concept that led to Walter Cannon's theory of physiological homeostasis (2); Alan Turing's model of pattern formation (3); as well as investigations of metabolic end-product inhibition (4), metabolic oscillations (5), and transcriptional self-repression (6). Biological feedback concepts were further influenced by chemical oscillation theories (7) and the field of cybernetics (8). It has more recently become appreciated that the concept of feedback may be useful as a framework for understanding how intracellular signaling systems elicit specific cell behavior.Mammalian species use over 3000 signaling proteins and over 15 second messengers to build hundreds of cell-specific signaling systems. Many of the signaling components have multiple upstream regulators and downstream targets, creating a web of connectivity within and between signaling pathways (9). The presence of multiple feedback loops in these systems (10) poses a challenge to understanding how receptor inputs control cellular behavior. We discuss how recurring feedback designs, or motifs (11), mediate biological functions such as bistability, oscillation, polarization, and robustness. Our goal was to generate a comprehensive guide for feedback in signal transduction that would also be instructive for understanding transcription networks, control of metabolism, pattern formation, the cell cycle, and the behavior of circadian oscillators.We focus on two mammalian signaling systems: the receptor-triggered Ca 2+ signaling system in nonexcitable cells (12) and the phosphoinositide 3-kinase (PI3K) signaling pathway in chemotactic neutrophils (13). These were chosen because of existing knowledge of feedback mechanisms that generate both simple and complex temporal and spatial signaling responses (Fig. 1). We use graphical representations of feedback motifs, with signaling components shown as vertices and directed negative and positive regulatory steps shown as arrows with and without minus symbols, respectively (Figs. 2 to 5, gray background). An arrow may consist of multiple steps so that a single positive arrow could reflect, for example, the net effect of two Supporting Online Materialwww.sciencemag

Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation.

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