Mechanism of Translational Control by the Fragile X Mental Retardation Protein and Creation of the FMRP CTAG Mouse

The Fragile X Mental Retardation Protein (FMRP) is a neuronal RNA-binding protein that is predominantly associated with polyribosomes. Loss of FMRP results in Fragile X Syndrome, characterized by mental retardation, autism and epilepsy. FMRP was recently found to be associated with a specific set of mRNAs with key roles in neuronal function and to physically interact with targeted mRNAs along their entire coding sequences (Darnell 2011). Here, we find that FMRP inhibits translation on these target transcripts by stalling ribosomes, both in vivo and in rabbit reticulocyte lysate programmed with endogenous brain polyribosomes. In these systems, loss of FMRP function resulted in increased ribosome runoff after treatment with puromycin, a drug that acts specifically on translocating ribosomes. In addition to genetic loss-of-function models, FMRPdependent relief of ribosome stalling could be induced by acute biochemical removal of FMRP from polysomes, indicating reversibility of stalling. FMRP was also directly visualized on stalled polysomes by immunoelectron microscopy. Together, these results suggest a model in which FMRP actively regulates translation of target mRNAs by stalling ribosomes. To further advance our understanding of FMRP function, we have created the FMRP cTAG mouse, a knock-in model in which FMRP can be conditionally tagged with AcGFP in a Cre-dependent manner while maintaining wt FMRP expression in all Cre-negative cells. The cTAG mouse will be a valuable tool in the study of cell type-specific FMRP function

Of mongooses and mitigation: ecological analogues to geoengineering

Anthropogenic global warming is a growing environmental problem resulting from unintentional human intervention in the global climate system. If employed as a response strategy, geoengineering would represent an additional intentional human intervention in the climate system, with the intent of decreasing net climate impacts. There is a rich and fascinating history of human intervention in environmental systems, with many specific examples from ecology of deliberate human intervention aimed at correcting or decreasing the impact of previous unintentionally created problems. Additional interventions do not always bring the intended results, and in many cases there is evidence that net impacts have increased with the degree of human intervention. In this letter, we report some of the examples in the scientific literature that have documented such human interventions in environmental systems, which may serve as analogues to geoengineering. We argue that a high degree of system understanding is required for increased intervention to lead to decreased impacts. Given our current level of understanding of the climate system, it is likely that the result of at least some geoengineering efforts would follow previous ecological examples where increased human intervention has led to an overall increase in negative environmental consequences

COX24 codes for a mitochondrial protein required for processing of the COX1 transcript

In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Localization and local translation of Arc/Arg3.1 mRNA at synapses: some observations and paradoxes.

Arc is a unique immediate early gene whose expression is induced as synapses are being modified during learning. The uniqueness comes from the fact that newly synthesized Arc mRNA is rapidly transported throughout dendrites where it localizes near synapses that were recently activated. Here, we summarize aspects of Arc mRNA translation in dendrites in vivo, focusing especially on features of its expression that are paradoxical or that donot fit in with current models of how Arc protein operates. Findings from in vivo studies that donot quite fit include: (1) Following induction of LTP in vivo, Arc mRNA and protein localize near active synapses, but are also distributed throughout dendrites. In contrast, Arc mRNA localizes selectively near active synapses when stimulation is continued as Arc mRNA is transported into dendrites; (2) Strong induction of Arc expression as a result of a seizure does not lead to a rundown of synaptic efficacy in vivo as would be predicted by the hypothesis that high levels of Arc cause glutamate receptor endocytosis and LTD. (3) Arc protein is synthesized in the perinuclear cytoplasm rapidly after transcriptional activation, indicating that at least a pool of Arc mRNA is not translationally repressed to allow for dendritic delivery; (4) Increases in Arc mRNA in dendrites are not paralleled by increases in levels of exon junction complex (EJC) proteins. These results of studies of mRNA trafficking in neurons in vivo provide a new perspective on the possible roles of Arc in activity-dependent synaptic modifications

Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies

ABSTRACTBiologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration–time curve (AUC0–672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL