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CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling 1 . SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes 2-5 . However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development ., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In a short hairpin RNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small guanosine triphosphatase (GTPase)-a protein previously implicated in endomembrane trafficking-as a regulator of the phosphatidylinositol 3'-OH kinase (PI3K) pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and copurifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive platelet-derived growth factor receptor α to LAMP2-positive endomembranes in the absence of ligand, suggesting that there may be latent oncogenic potential in dysregulated endomembrane trafficking., (Copyright © 2015, American Association for the Advancement of Science.)

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.

Background: Image-based screens can produce hundreds of measured features for each of hundreds of millions of individual cells in a single experiment., Results: Here, we describe CellProfiler Analyst, open-source software for the interactive exploration and analysis of multidimensional data, particularly data from high-throughput, image-based experiments., Conclusion: The system enables interactive data exploration for image-based screens and automated scoring of complex phenotypes that require combinations of multiple measured features per cell.

The recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.

RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.

The Ca(2+) channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca(2+) channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. alpha(1A)-Subunits are thought to support both P- and Q-type Ca(2+) channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca(2+) delivery system at excitatory nerve terminals. We generated alpha(1A)-deficient mice (alpha(1A)(-/-)) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca(2+) channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca(2+) entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A).

Objective: To determine the relative importance of medical and nonmedical factors influencing generalists' decisions to refer, and of the factors that might avert unnecessary referrals., Design: Prospective survey of all referrals from generalists to subspecialists over a 5-month period., Setting: University hospital outpatient clinics., Participants: Fifty-seven staff physicians in general internal medicine, family medicine, dermatology, orthopedics, gastroenterology, and rheumatology., Measurements and Main Results: For each referral, the generalist rated a number of medical and nonmedical reasons for referral, as well as factors that may have helped avert the referral; the specialist seeing the patient then rated the appropriateness, timeliness, and complexity of the referral. Both physicians rated the potential avoidability of the referral by telephone consultation. Generalists were influenced by a combination of both medical and nonmedical reasons for 76% of the referrals, by only medical reasons in 20%, and by only nonmedical reasons in 3%. In 33% of all referrals, generalists felt that training in simple procedures or communication with a generalist or specialist colleague would have allowed them to avoid referral. Specialists felt that the vast majority of referrals were timely (as opposed to premature or delayed) and of average complexity. Although specialists rated most referrals as appropriate, 30% were rated as possibly appropriate or inappropriate. Generalists and specialists failed to agree on the avoidability of 34% of referrals., Conclusions: Generalists made most referrals for a combination of medical and nonmedical reasons, and many referrals were considered avoidable. Increasing procedural training for generalists and enhancing informal channels of communication between generalists and subspecialists might result in more appropriate referrals at lower cost.

Several types of calcium channels found in the central nervous system are possible participants in triggering neurotransmitter release. Synaptic transmission between hippocampal CA3 and CA1 neurons was mediated by N-type calcium channels, together with calcium channels whose pharmacology differs from that of L- and P-type channels but resembles that of the Q-type channel encoded by the alpha 1A subunit gene. Blockade of either population of channels strongly increased enhancement of synaptic transmission with repetitive stimuli. Even after complete blockade of N-type channels, transmission was strongly modulated by stimulation of neurotransmitter receptors or protein kinase C. These findings suggest a role for alpha 1A subunits in synaptic transmission and support the idea that neurotransmitter release may depend on multiple types of calcium channels under physiological conditions.

We have used the techniques of microdialysis and in vivo voltammetry to monitor striatal dopamine and ascorbate, as well as motor activity in unanaesthetized, freely-moving rats. Systemic administration of the non-selective dopamine receptor agonist apomorphine (0.5 mg/kg, s.c.) caused a decrease in dopamine, an increase in ascorbate, stereotyped behaviour and a generalized increase in motor activity. Separate systemic applications of the D1 receptor agonist SKF 38393 (10 mg/kg, s.c.) and the D2 receptor agonist Quinpirole (0.1 mg/kg s.c.) caused a decrease in dopamine but had no effect on ascorbate or motor activity. After coadministration of these drugs, there was an increase in both ascorbate and motor activity. Local application of apomorphine (0.01 mM) caused a reduction in dopamine similar to that seen following systemic application but had no effect on ascorbate or motor activity. The present results demonstrate that dopamine, via D1 and D2 receptors outside the striatum, plays an important role in the control of ascorbate release. These results lend further support to the hypothesis that changes in ascorbate levels are an index of glutamatergic neurotransmission.