Dissecting eukaryotic translation and its control by ribosome density mapping

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5′ and 3′ ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo

CytoCensus, mapping cell identity and division in tissues and organs using machine learning.

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues

Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background: The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported. Methodology/Principal Findings: In the present study we found that asc1-deletion mutant (asc1D) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1D cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells ’ survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1D/pkc1D) to cell-wall stress conditions, and high basal level of PKC signaling in asc1D. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions. Conclusions/Significance: Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p act

mRNA association by aminoacyl tRNA synthetase occurs at a putative anticodon mimic and autoregulates translation in response to tRNA levels.

Aminoacyl-tRNA synthetases (aaRSs) are well studied for their role in binding and charging tRNAs with cognate amino acids. Recent RNA interactome studies had suggested that these enzymes can also bind polyadenylated RNAs. Here, we explored the mRNA repertoire bound by several yeast aaRSs. RNA immunoprecipitation (RIP) followed by deep sequencing revealed unique sets of mRNAs bound by each aaRS. Interestingly, for every tested aaRSs, a preferential association with its own mRNA was observed, suggesting an autoregulatory process. Self-association of histidyl-tRNA synthetase (HisRS) was found to be mediated primarily through binding to a region predicted to fold into a tRNAHis anticodon-like structure. Introducing point mutations that are expected to disassemble this putative anticodon mimic alleviated self-association, concomitant with increased synthesis of the protein. Finally, we found that increased cellular levels of uncharged tRNAHis lead to reduced self-association and increased HisRS translation, in a manner that depends on the anticodon-like element. Together, these results reveal a novel post-transcriptional autoregulatory mechanism that exploits binding mimicry to control mRNA translation according to tRNA demands

Yeast: Translation Regulation and Localized Translation

Translation regulation and localized translation are essential for protein synthesis, controlling all aspects of cellular function in health and disease [...

Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9-2

<p><b>Copyright information:</b></p><p>Taken from "Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Genomics 2007;8():285-285.</p><p>Published online 21 Aug 2007</p><p>PMCID:PMC2020489.</p><p></p> collected from wild-type or strains and mixed with RNase H and ODN complementary to the region indicated by an arrow on the schematic presentation of each mRNA. This should lead to a cleavage of the mRNA at the region complementary to the ODN and to result in two fragments: 5' fragment (depicted in black) and 3' fragment (depicted in white). Following the RNase H cleavage reaction, samples were separated on a sucrose gradient into 18 fractions and subjected to northern analysis. Hybridization for Yhb1 (A) was performed using a probe that recognizes the entire open reading frame, therefore both fragments appear in the same panel. Hybridization for YGR026W (B) was performed using probes specific either to the 5' or 3' fragments of the mRNA (upper and lower panels, respectively). Arrows to the left of each panel indicate the migration position of the cleavage product, as well as residual uncut mRNA ("full-length"). Histograms represent the quantitation results of the 5' fragment (black bars) and 3' fragment (white bars) of each mRNA

Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9-1

<p><b>Copyright information:</b></p><p>Taken from "Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Genomics 2007;8():285-285.</p><p>Published online 21 Aug 2007</p><p>PMCID:PMC2020489.</p><p></p>parated on formaldehyde-agarose gel and subjected to northern blotting. Three blots were prepared, one from each experimental repeat (indicated by bars at the left). Blots were hybridized with probes complementary to the genes indicated at left of each panel and with a probe for the spiked-in Phe RNA. B) Comparison of quantitation results of the northern analysis with microarray data. Black bars represent the northern analysis signal from each fraction normalized to its corresponding signal of the Phe RNA and calculated as a percent of total signal of that mRNA in the gradient. Open bars represent the ratio obtained in the microarray analysis (note that the histogram has two Y-axes) normalized by a signal from the transcribed mRNAs. Fractions where open bars are missing indicate spots that did not pass the quality criteria in the microarray assay. Note that the Y-axis scale of the microarray results differs from gene to gene. This is probably due to differences in their mRNAs abundances compared to the reference sample

Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9-0

<p><b>Copyright information:</b></p><p>Taken from "Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Genomics 2007;8():285-285.</p><p>Published online 21 Aug 2007</p><p>PMCID:PMC2020489.</p><p></p>phase and harvested. Cell lysates were separated on a 10%–50% sucrose gradient and the OD254 along the gradient was monitored. The sedimentation position of ribosomal complexes (40S, 60S, 80S and polysomes) is indicated on each panel

Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9-5

<p><b>Copyright information:</b></p><p>Taken from "Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Genomics 2007;8():285-285.</p><p>Published online 21 Aug 2007</p><p>PMCID:PMC2020489.</p><p></p>phase and harvested. Cell lysates were separated on a 10%–50% sucrose gradient and the OD254 along the gradient was monitored. The sedimentation position of ribosomal complexes (40S, 60S, 80S and polysomes) is indicated on each panel