Identifying pathogenicity of human variants via paralog-based yeast complementation

Yang, Fan; Sun, Song; Tan, Guihong; Costanzo, Michael; Hill, David E.; Vidal, Marc; Andrews, Brenda; Boone, Charles; Roth, Frederick P.

doi:10.1371/journal.pgen.1006779

Cited by 32 publications

(40 citation statements)

References 56 publications

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“…Following the results of our computational analysis, we searched for a phase-separating protein candidate containing RBDs and 1 PrLD (Figure 2A and 2B). To this aim, we focused on S. cerevisiae that (i) has been widely validated as a model to study phase separation 12,19,52 , (ii) produces abundant prion-like proteins 29,46 and (iii) has membraneless organelles and protein quality control system conserved with high eukaryotes 53,54,55 .…”

Section: A Model To Monitor Changes In Phase Separationmentioning

confidence: 99%

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Gotor

Armaos

Calloni

et al. 2020

Preprint

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Biomolecular condensates are membrane-less organelles mainly composed of proteins and RNAs that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to phase separate is encoded in their sequences, yet it is still unknown which domains drive the process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting biomolecular condensation. Using advanced computational models to predict the phase separation propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-Binding Domains (RBDs). We found that while just one PrLD is sufficient to drive protein condensation, multiple RBDs are needed to modulate the dynamicity of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.

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Section: A Model To Monitor Changes In Phase Separationmentioning

confidence: 99%

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Gotor

Armaos

Calloni

et al. 2020

Preprint

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“…72 expression profiles) rather than direct functional assays. However, recent systematic studies of 73 functional replacement of yeast genes by their orthologs from humans [18][19][20][21] and bacteria [22] 74 demonstrate the power of inter-species gene swaps to directly test functional divergence and 75 identify properties that determine functional conservation across vast evolutionary distances. Here, 76we sought to investigate the functional divergence across gene families by directly swapping 77 human genes belonging to extended families into yeast.…”

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confidence: 99%

Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Laurent

Garge

et al. 2019

Preprint

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1Despite over a billion years of evolutionary divergence, several thousand human genes possess 2 clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in 3 one or both lineages. The ortholog conjecture postulates that orthologous genes between species 4 retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free 5 to diverge in function. However, the retention of ancestral functions among co-orthologs between 6 species and within gene families has been difficult to test experimentally at scale. In order to 7 investigate how ancestral functions are retained or lost post-duplication, we systematically 8 replaced hundreds of essential yeast genes with their human orthologs from gene families that have 9 undergone lineage-specific duplications, including those with single duplications (one yeast gene 10 to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a 11 variable pattern of replaceability across different ortholog classes, with an obvious trend towards 12 differential replaceability inside gene families, rarely observing replaceability by all members of 13 a family. We quantify the ability of various properties of the orthologs to predict replaceability, 14showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and 15 tissue-specific expression of the human co-orthologs, i.e. the human proteins that are less diverged 16 from their yeast counterpart and more ubiquitously expressed across human tissues more often 17 replace their single yeast ortholog. These trends were consistent with in silico simulations 18demonstrating that when only one ortholog is replaceable, it tends to be the least diverged of the 19 pair. Replaceability of yeast genes having more than two human co-orthologs was marked by 20 retention of orthologous interactions in functional or protein networks as well as by more ancestral 21 subcellular localization. Overall, we performed >400 human gene replaceability assays revealing 22 56 new human-yeast complementation pairs, thus opening up avenues to further functionally 23 characterize these human genes in a simplified organismal context. 24 25 and are distinguished from those related by speciation, termed orthologs [8,9]. Importantly, an 46 expanded family of paralogs in one lineage may be co-orthologs to a single gene in another lineage. 47How ancestral functions are partitioned, lost, or retained between paralogs and orthologs during 48 these duplication events is a major topic of study for evolutionary biology. To address these 49 questions, the ortholog conjecture was put forth, a major thesis stating that orthologs are more 50 likely to retain function between species than paralogs, which will tend to diverge in function after 51 duplication due to drift and relaxed selection [10,11]. This conjecture underlies common methods 52 by which functions are assigned to newly discovered or understudied genes in un-annotated 53 4 genomes ...

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“…The PTEN gene encodes a PtdIns (3,4,5)P3 phosphatase which acts on the 3' phosphate group of the inositol ring [13]. PTEN does not have a direct yeast homolog and its usual substrate PtdIns (3,4,5)P3 is not present in yeast. PTEN overexpression did not affect growth of wt yeast ( Fig S1A), suggesting yeast physiology is sufficiently robust to buffer against PTEN action in yeast.…”

Section: Synthetic Dosage Lethality Screening To Identify Yeast Straimentioning

confidence: 99%

“…Variants of the gene may then be assayed by the degree to which they achieve this (Fig 1A), the assumption being that non-functional variants will fail to rescue growth. This approach has also been extended to paralogous genes [5].…”

Section: Introductionmentioning

confidence: 99%

“…However, in cases where there is neither a yeast orthologue of the human gene, or expression of the human gene in yeast does not lead to a phenotype, then there is a requirement for a different type of assay. Indeed, fewer than 30% of human disease genes have a direct yeast orthologue [5]. Here, using the specific example of the human PTEN gene, we described a general approach that can be used to identify yeast strains that can be used to measure the functionality of disease gene variants, a technique we name Sentinel Interaction Mapping (SIM; Fig 1C).…”

Section: Introductionmentioning

confidence: 99%

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Sentinel Interaction Mapping (SIM) – A generic approach for the functional analysis of human disease gene variants using yeast

Young

Post

Chao

et al. 2020

Preprint

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Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using Synthetic Dosage Lethality screening, "sentinel" yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in high-throughput fashion through simple growth assays using either solid or liquid media.Sentinel Interaction Mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

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Identifying pathogenicity of human variants via paralog-based yeast complementation

Cited by 32 publications

References 56 publications

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Sentinel Interaction Mapping (SIM) – A generic approach for the functional analysis of human disease gene variants using yeast

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