The rockefeller university

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2(KLHDC10) E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.

The MED1 subunit has been shown to mediate ligand-dependent binding of the Mediator coactivator complex to multiple nuclear receptors, including the adipogenic PPAR gamma, and to play an essential role in ectopic PPAR gamma-induced adipogenesis of mouse embryonic fibroblasts. However, the precise roles of MED1, and its various domains, at various stages of adipogenesis and in adipose tissue have been unclear. Here, after establishing requirements for MED1, including specific domains, for differentiation of 3T3L1 cells and both primary white and brown preadipo-cytes, we used multiple genetic approaches to assess requirements for MED1 in adipocyte formation, maintenance, and function in mice. We show that MED1 is indeed essential for the differentiation and/or function of both brown and white adipocytes, as its absence in these cells leads to, respectively, defective brown fat function and lipodys-trophy. This work establishes MED1 as an essential transcriptional coactivator that ensures homeostatic functions of adipocytes.

Background Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells. Methods Autoantibodies in the plasma of pSLE patients (n = 30), healthy children (n = 37), and disease controls juvenile idiopathic arthritis (JIA) (n = 32) and juvenile dermatomyositis (JDM) (n = 60), were measured by ELISA. Expression of p40 in immune cells was assessed by flow cytometry. Markers of neutrophil activation and death were quantitated by ELISA. Results IgG and IgA autoantibodies reactive with p40 were detected in the pSLE patients, but were low in healthy controls and in JIA or JDM. pSLE patients with active disease (13 of them newly diagnosed) had higher titers than the same patients after effective therapy (p = 0.0003). IgG titers correlated with SLEDAI (r = 0.65, p = 0.0001), ESR (r = 0.43, p = 0.02), and anti-dsDNA antibodies (r = 0.49, p < 0.03), and inversely with complement C3 (r = -0.55, p = 0.002) and C4 (r = -0.51, p = 0.006). p40 protein was detected in a subpopulation of CD66b(+) granulocytes in pSLE, as well as in adult SLE patients. Myeloperoxidase and neutrophil elastase complexed with DNA and the neutrophil-derived S100A8/A9 were elevated in plasma from pSLE patients with active disease and correlated with anti-p40 autoantibodies and disease activity. Conclusions Children with active SLE have elevated IgG and IgA autoantibodies against L1 p40, and this protein can be detected in circulating granulocytes in both pediatric and adult SLE patients. P40 expression and autoantibody levels correlate with disease activity. Markers of neutrophil activation and death also correlate with these autoantibodies and with disease activity, suggesting that neutrophils express L1 and are a source of p40.

Comprehensive descriptions of animal behavior require precise three-dimensional (3D) measurements of whole-body movements. Although two-dimensional approaches can track visible landmarks in restrictive environments, performance drops in freely moving animals, due to occlusions and appearance changes. Therefore, we designed DANNCE to robustly track anatomical landmarks in 3D across species and behaviors. DANNCE uses projective geometry to construct inputs to a convolutional neural network that leverages learned 3D geometric reasoning. We trained and benchmarked DANNCE using a dataset of nearly seven million frames that relates color videos and rodent 3D poses. In rats and mice, DANNCE robustly tracked dozens of landmarks on the head, trunk, and limbs of freely moving animals in naturalistic settings. We extended DANNCE to datasets from rat pups, marmosets, and chickadees, and demonstrate quantitative profiling of behavioral lineage during development.

Repeat sequences account for over half of the human genome and represent a significant source of variation that underlies physiological and pathological states. Yet, their study has been hindered due to limitations in shortreads sequencing technology and difficulties in assembly. A important category of repetitive DNA in the human genome is comprised of tandem repeats (TRs), where repetitive units are arranged in a head-to-tail pattern. Compared to other regions of the genome, TRs carry between 10 and 10,000 fold higher mutation rate. There are several mutagenic mechanisms that can give rise to this propensity toward instability, but their precise contribution remains speculative. Given the high degree of homology between these sequences and their arrangement in tandem, once damaged, TRs have an intrinsic propensity to undergo aberrant recombination with non-allelic exchange and generate harmful rearrangements that may undermine the stability of the entire genome. The dynamic mutagenesis at TRs has been found to underlie individual polymorphism associated with neurodegenerative and neuromuscular disorders, as well as complex genetic diseases like cancer and diabetes. Here, we review our current understanding of the surveillance and repair mechanisms operating within these regions, and we describe how alterations in these protective processes can readily trigger mutational signatures found at TRs, ultimately resulting in the pathological correlation between TRs instability and human diseases. Finally, we provide a viewpoint to counter the detrimental effects that TRs pose in light of their selection and conservation, as important drivers of human evolution.

Connectionist and dynamic field models consist of a set of coupled first-order differential equations describing the evolution in time of different units. We compare three numerical methods for the integration of these equations: the Euler method, and two methods we have developed and present here: a modified version of the fourth-order Runge Kutta method, and one semi-analytical method. We apply them to solve a well-known nonlinear connectionist model of retrieval in single-digit multiplication, and show that, in many regimes, the semi-analytical and modified Runge Kutta methods outperform the Euler method, in some regimes by more than three orders of magnitude. Given the outstanding difference in execution time of the methods, and that the EM is widely used, we conclude that the researchers in the field can greatly benefit from our analysis and developed methods.

Several OPRD1 intronic variants were associated with opioid addiction (OD) in a population-specific manner. This follow-up study aims to further characterize the OPRD1 haplotype pattern of the risk variants in different populations and apply in silico analysis to identify potential causal variants. A population-specific haplotype pattern was revealed based on six OPRD1 eQTL SNPs and five common haplotypes were identified in a sample of European ancestry (CEU). A European-specific haplotype ('Hap 3') that includes SNPs previously associated with OD and is tagged by SNP rs2236861 is more common in subjects with OD. It is quite common (10%) in CEU but is absent in the African sample (YRI) and extends upstream of OPRD1. SNP rs2236857 is most probably a non-causal variant in LD with the causal SNP/s in a population-specific manner. The study provides an explanation for the lack of association in African Americans, despite its high frequency in this population. OD samples homozygous for 'Hap 3' were reanalyzed using a denser coverage of the region and revealed at least 25 potentially regulatory SNPs in high LD. Notably, GTEx data indicate that some of the SNPs are eQTLs for the upstream phosphatase and actin regulator 4 (PHACTR4), in the cortex, and others are eQTLs for OPRD1 and the upstream lncRNA ENSG00000270605, in the cerebellum. The study highlights the limitation of single SNP analysis and the sensitivity of association studies of OPRD1 to a genetic background. It proposes a long-range functional connection between OPRD1 and PHACTR4. PHACTR4, a mediator of cytoskeletal dynamics, may contribute to drug addiction by modulating synaptic plasticity.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 30 UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

Interspecific hybridization is a stressful condition that can lead to sterility and/or inviability through improper gene regulation in Drosophila species with a high divergence time. However, the extent of these abnormalities in hybrids of recently diverging species is not well known. Some studies have shown that in Drosophila, the mechanisms of postzygotic isolation may evolve more rapidly in males than in females and that the degree of viability and sterility is associated with the genetic distance between species. Here, we used transcriptomic comparisons between two Drosophila mojavensis subspecies and D. arizonae (repleta group, Drosophila) and identified greater differential gene expression in testes than in ovaries. We tested the hypothesis that the severity of the interspecies hybrid phenotype is associated with the degree of gene misregulation. We showed limited gene misregulation in fertile females and an increase in the amount of misregulation in males with more severe sterile phenotypes (motile vs. amotile sperm). In addition, for these hybrids, we identified candidate genes that were mostly associated with spermatogenesis dysfunction.