Activity

Different subsets of the tRNA pool in human cells are expressed in different cellular conditions. The ‘proliferation-tRNAs’ are induced upon normal and cancerous cell division, while the ‘differentiation-tRNAs’ are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. read more We measured tRNA essentiality for cellular growth and found that most proliferation-tRNAs are essential compared to differentiation- tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation-tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.The prefrontal cortex (PFC)’s functions are thought to include working memory, as its activity can reflect information that must be temporarily maintained to realize the current goal. We designed a flexible spatial working memory task that required rats to navigate – after distractions and a delay – to multiple possible goal locations from different starting points and via multiple routes. This made the current goal location the key variable to remember, instead of a particular direction or route to the goal. However, across a broad population of PFC neurons, we found no evidence of current-goal-specific memory in any previously reported form – that is differences in the rate, sequence, phase, or covariance of firing. This suggests that such patterns do not hold working memory in the PFC when information must be employed flexibly. Instead, the PFC grouped locations representing behaviorally equivalent task features together, consistent with a role in encoding long-term knowledge of task structure.Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil – carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited because they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme-substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When we exposed populations of Drosophila melanogaster to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, we found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive.The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as TC doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.