Inference of single-cell phylogenies from lineage tracing data using Cassiopeia.

The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 50 UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.UCSF Program for Breakthrough Biomedical Research - Sandler FoundationNIH DP5OD017895, GM061835Burroughs Wellcome FundDavid and LucilePackard FoundationUS Department of the Interior Grant D12AP00025US Army Research Office W911NF-12-1-0552Cellular and Molecular Biolog

Local Correlations in Codon Preferences Do Not Support a Model of tRNA Recycling

It has been proposed that patterns in the usage of synonymous codons provide evidence that individual tRNA molecules are recycled through the ribosome, translating several occurrences of the same amino acid before diffusing away. The claimed evidence is based on counting the frequency with which pairs of synonymous codons are used at nearby occurrences of the same amino acid, as compared to the frequency expected if each codon were chosen independently from a single genome-wide distribution. We show that such statistics simply measure variation in codon preferences across a genome. As a negative control on the potential contribution of pressure to exploit tRNA recycling on these signals, we examine correlations in the usage of codons that encode different amino acids. We find that these controls are statistically as strong as the claimed evidence and conclude that there is no informatic evidence that tRNA recycling is a force shaping codon usage

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics

CRISPR-based functional genomics in human dendritic cells

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of &gt;94% across &gt;300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.</jats:p

An open 3D CFD model for the investigation of flow environments experienced by freshwater fish

Computational fluid dynamics (CFD) provides a powerful numerical tool to simulate and study many of the complex fluid-body interactions experienced by freshwater fish. However, major gaps remain in the application of CFD to study the fluid-body interactions of fish, including the absence of an openly available reference body geometry, the lack of a detailed study on suitable numerical methods and a deficit of available velocity laboratory measurements for model calibration and validation. To address these gaps, we provide a set of numerical models based on the open-source CFD toolkit OpenFOAM. The contributions of this work are two-fold: First, to provide a validated openly available numerical setup using a realistic fish model geometry including laboratory velocity measurements. Second, to determine the best-performing turbulence models and near-wall treatments using Reynolds-Averaged Navier-Stokes (RANS) numerical simulations. Finally, we conclude with a critical evaluation of the effects and trade-offs of resolving or modelling the boundary layer (BL) in numerical studies of fish-shaped bodie

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′ UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.National Institutes of Health (U.S.) (Grant GM061835

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates

Changes in tRNA binding site enrichments between a pair of experiments with and without CHX are matched by areas of downstream waves in the CHX experiment.

<p>For a pair of experiments with and without CHX by Jan [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005732#pgen.1005732.ref041" target="_blank">41</a>], the sum of each codon identity’s changes in mean relative enrichment at the A-, P-, and E-sites between the two experiments (green area in insets) is plotted against the total excess or deficit of enrichment in the CHX experiment from 6 to 65 codons downstream (red area in insets). The area of each codon identity’s downstream peak is strongly predicted by changes in enrichment at the tRNA binding sites, consistent with the hypothesis that downstream peaks are transient waves caused by continued elongation with disrupted dynamics in the presence of CHX.</p