Biology subjects

Landry, C. R.

Publications and source records attributed to Landry, C. R..

44 records · Page 3Linked to original sources

bioRxiv2020

The specificity of SRC Homology 3 (SH3) domain interactions is modulated by their protein context in vivo

Protein-protein interactions (PPIs) between modular binding domains and their target peptide motifs are thought to largely depend on the intrinsic binding specificities of the domains. By combining deletion, mutation, swapping and shuffling of SRC Homology 3 (SH3) domains and measuring their impact on protein interactions, we find that most SH3s do not autonomously dictate PPI specificity in vivo. The identity of the host protein and the position of the SH3 domains within their host are both critical for PPI specificity, for cellular functions and for key biophysical processes such as phase separation. Our work demonstrates the importance of the interplay between a modular PPI domain such as SH3 and its host protein in establishing specificity to wire PPI networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2019

Systematic perturbation of yeast essential genes using base editing

Base editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of an editor based on a cytidine deaminase, the Target-AID base editor, to systematically modify genes genome-wide using the set of yeast essential genes. We tested the effect of mutating around 17,000 individual sites in parallel across more than 1,500 genes in a single experiment. We identified over 1,100 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we predicted the protein variants caused by each of these gRNAs. We found that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious by independent methods based on site conservation and predicted protein destabilization. Finally, we identify key features to design effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.

synthetic biology↗

bioRxiv2019

Paralog dependency indirectly affects the robustness of human cells

Gene duplicates provide protection against loss-of-function mutations. This protective redundancy partly relies on the fact that paralogs carry their functions independently, i.e. the inactivation of one gene copy does not impair the function of the other copy. However, a significant fraction of paralogous proteins may form functionally dependent pairs, for instance through heteromerization. As a consequence, one could expect these heteromeric paralogs to be less protective against deleterious mutations. To test this hypothesis, we examined the fitness landscape of gene loss-of-function by CRISPR-Cas9 in more than 450 human cell lines. Our analysis revealed a robustness landscape of human cells showing regions of higher vulnerability to gene inactivation as a function of key paralog properties. We find that heteromerizing paralogs are indeed less protective than non-heteromeric ones, but this association is largely due to their higher abundance and their larger number of protein-protein interaction partners.

evolutionary biology↗

bioRxiv2018

beditor: A computational workflow for designing libraries of guide RNAs for CRISPR base editing

CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, PAM recognition sequences and genomes of many species. Additionally, in order to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates, called beditor scores are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: editable base falls within maximum activity window of the CRISPR-mediated base editor and produces non-confounding mutational effects with minimal off-target effects. We demonstrate the utility of the software by designing guide RNAs for base-editing mediated edition of thousands of clinically important human disease mutations.

synthetic biology↗

bioRxiv2018

The high turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations

Little is known about the rate of emergence of genes de novo, how they spread in populations and what their initial properties are. We examined wild yeast (Saccharomyces paradoxus) populations to characterize the diversity and turnover of intergenic ORFs over short evolutionary time-scales. With ~34,000 intergenic ORFs per individual genome for a total of ~64,000 orthogroups identified, we found de novo ORF formation to have a lower estimated turnover rate than gene duplication. Hundreds of intergenic ORFs show translation signatures similar to canonical genes. However, they have lower translation efficiency, which could reflect a mechanism to reduce their production cost or simply a lack of optimization. We experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Translated intergenic ORFs tend to display low expression levels with sequence properties that generally are close to expectations based on intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared less than 110 Kya ago, already show gene- like characteristics, suggesting that the raw material for functional innovations could appear over short evolutionary time-scales.

evolutionary biology↗

bioRxiv2018

Differences between the de novo proteome and its non-functional precursor can result from neutral constraints on its birth process, not necessarily from natural selection alone

Proteins are among the most important constituents of biological systems. Because all proteins ultimately evolved from previously non-coding DNA, the properties of these non-coding sequences and how they shape the birth of novel proteins are also expected to influence the organization of biological networks. When trying to explain and predict the properties of novel proteins, it is of particular importance to distinguish the contributions of natural selection and other evolutionary forces. Studies in the field typically use non-coding DNA and GC-content-based random-sequence models to generate random expectations for the properties of novel functional proteins. Deviations from these expectations have been interpreted as the result of natural selection. However, interpreting such deviations requires a yet-unattained understanding of the raw material of de novo gene birth and its relation to novel functional proteins. We mathematically show how the importance of the \"junk\" polypeptides that make up this raw material goes beyond their average properties and their filtering by natural selection. We find that the mean of any property among novel functional proteins also depends on its variance among junk polypeptides and its correlation with their rate of evolutionary turnover. In order to exemplify the use of our general theoretical results, we combine them with a simple model that predicts the means and variances of the properties of junk polypeptides from the genomic GC content alone. Under this model, we predict the effect of GC content on the mean length and mean intrinsic disorder of novel functional proteins as a function of evolutionary parameters. We use these predictions to formulate new evolutionary interpretations of published data on the length and intrinsic disorder of novel functional proteins. This work provides a theoretical framework that can serve as a guide for the prediction and interpretation of past and future results in the study of novel proteins and their properties under various evolutionary models. Our results provide the foundation for a better understanding of the properties of cellular networks through the evolutionary origin of their components.

evolutionary biology↗

bioRxiv2018

Highly efficient CRISPR gene editing in yeast enabled by double selection

CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

synthetic biology↗

bioRxiv2017

Deep transcriptome annotation suggests that small and large proteins encoded in the same genes often cooperate

Recent studies in eukaryotes have demonstrated the translation of alternative open reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and evolutionary patterns indicate that altORFs are particularly constrained in CDSs that evolve slowly. Thousands of predicted alternative proteins are detected in proteomic datasets by reanalysis with a database containing predicted alternative proteins. Protein domains and co-conservation analyses suggest potential functional cooperation or shared function between small and large proteins encoded in the same genes. This is illustrated with specific examples, including altMID51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many coding genes code for more than one protein that are often functionally related.

biochemistry↗