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Jonathan S Weissman

Publications and source records attributed to Jonathan S Weissman.

At least 19 recordsLinked to original sources
PubMed2026

Expanding the human proteome with microproteins and peptideins.

A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains of what has been missed in previous analyses. Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states1-3, with major implications for biomedical science. However, a key gap in knowledge has been which ncORFs produce small microproteins or alternative protein molecules that contribute to the human proteome. Here we report the collaborative efforts of the TransCODE Consortium4 to produce a consensus landscape of protein-level evidence for ncORFs. We show that about 25% of a set of 7,264 ncORFs gives rise to detectable peptides in a large-scale analysis of 95,520 proteomics experiments. We develop an annotation framework for ncORF-encoded microproteins as human proteins and codify the new conceptual model of 'peptideins' as microproteins that have indeterminate potential as functional proteins. To probe the biological implications of peptideins, we create an evolutionary analysis approach, termed ORF relative branch length (ORBL), and determine that evolutionary constraint is common and associates with observation of ncORF-derived peptides. We then characterize a pan-essential cellular phenotype for one peptidein from the OLMALINC long non-coding RNA. Overall, we generate public research tools supported by GENCODE and PeptideAtlas and advance biomedical discovery for understudied components of the human proteome.

Humans↗
PubMed2025

Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.

Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.

Humans↗
PubMed2025

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins.

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

GENCODE↗
PubMed2024

CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation and inflammatory signaling.

Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

RNA, Long Noncoding↗
PubMed2007

Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.

Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.

Cluster Analysis↗
PubMed2006

A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.

How the ER-associated degradation (ERAD) machinery accurately identifies terminally misfolded proteins is poorly understood. For luminal ERAD substrates, this recognition depends on their folding and glycosylation status as well as on the conserved ER lectin Yos9p. Here we show that Yos9p is part of a stable complex that organizes key components of ERAD machinery on both sides of the ER membrane, including the transmembrane ubiquitin ligase Hrd1p. We further demonstrate that Yos9p, together with Kar2p and Hrd3p, forms a luminal surveillance complex that both recruits nonnative proteins to the core ERAD machinery and assists a distinct sugar-dependent step necessary to commit substrates for degradation. When Hrd1p is uncoupled from the Yos9p surveillance complex, degradation can occur independently of the requirement for glycosylation. Thus, Yos9p/Kar2p/Hrd3p acts as a gatekeeper, ensuring correct identification of terminally misfolded proteins by recruiting misfolded forms to the ERAD machinery, contributing to the interrogation of substrate sugar status, and preventing glycosylation-independent degradation.

Amino Acid Sequence↗
PubMed2006

Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response.

The unfolded protein response (UPR) allows the endoplasmic reticulum (ER) to recover from the accumulation of misfolded proteins, in part by increasing its folding capacity. Inositol-requiring enzyme-1 (IRE1) promotes this remodeling by detecting misfolded ER proteins and activating a transcription factor, X-box-binding protein 1, through endonucleolytic cleavage of its messenger RNA (mRNA). Here, we report that IRE1 independently mediates the rapid degradation of a specific subset of mRNAs, based both on their localization to the ER membrane and on the amino acid sequence they encode. This response is well suited to complement other UPR mechanisms because it could selectively halt production of proteins that challenge the ER and clear the translocation and folding machinery for the subsequent remodeling process.

Amino Acid Sequence↗
PubMed2006

The physical basis of how prion conformations determine strain phenotypes.

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.

Amyloid↗
PubMed2006

Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.

A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.

Culture Media↗
PubMed2006

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Biological Evolution↗
PubMed2006

An efficient protein transformation protocol for introducing prions into yeast.

Although a range of robust techniques exists for transforming organisms with nucleic acids, approaches for introducing proteins into cells are far less developed. Here we describe a facile and highly efficient protein transformation protocol suitable for introducing prion particles, produced in vitro from pure protein or purified from an in vivo source, into yeast. Prion particles composed of amyloid forms of fragments of Sup35p, the protein determinant of the yeast prion state [PSI(+)], lead to dose-dependent de novo induction of [PSI(+)] with efficiencies approaching 100% at high protein concentrations. We also describe a procedure for generating distinct, self-propagating amyloid conformations of a prionogenic Sup35p fragment termed Sup-NM. Remarkably, infection of yeast with different Sup-NM amyloid conformations leads to distinct [PSI(+)] prion strains, establishing that the heritable differences in prion strain differences result directly from self-propagating differences in the conformations of the infectious protein. This protein transformation protocol can be readily adapted to the analysis of other yeast prion states, as well as to test the infectious (prion) nature of protein extracts from less well-characterized epigenetic traits. More generally, the protein transformation procedure makes it possible to bridge in vitro and in vivo studies, thus greatly facilitating efforts to explain the structural and mechanistic basis of prion inheritance.

Prions↗
PubMed2006

A strategy for extracting and analyzing large-scale quantitative epistatic interaction data.

Recently, approaches have been developed for high-throughput identification of synthetic sick/lethal gene pairs. However, these are only a specific example of the broader phenomenon of epistasis, wherein the presence of one mutation modulates the phenotype of another. We present analysis techniques for generating high-confidence quantitative epistasis scores from measurements made using synthetic genetic array and epistatic miniarray profile (E-MAP) technology, as well as several tools for higher-level analysis of the resulting data that are greatly enhanced by the quantitative score and detection of alleviating interactions.

Epistasis, Genetic↗
PubMed2005

Cotranscriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex.

The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.

Acetylation↗
PubMed2005

Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation.

Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate. In contrast, we found that in the context of full-length PDI, there is an asymmetry in the rate of oxidation of the two active sites. This asymmetry is the result of a dual effect: an enhanced rate of oxidation of the second catalytic (A') domain and the substrate-mediated inhibition of oxidation of the first catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide isomerase and disulfide oxidase, respectively. These findings reveal how native disulfide folding is accomplished in the endoplasmic reticulum and provide a context for understanding the proliferation of PDI homologs with combinatorial arrangements of thioredoxin domains.

Binding Sites↗
PubMed2005

Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.

We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.

Cluster Analysis↗
PubMed2005

Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen.

ER-associated degradation (ERAD) of glycoproteins depends on dual recognition of protein misfolding and remodeling of the substrate's N-linked glycans. After recognition, substrates are retrotranslocated to the cytosol for proteasomal degradation. To explore the directionality of this process, we fused a highly stable protein, DHFR, to the N or C terminus of the soluble ERAD substrate CPY* in yeast. Degradation of the C-terminal CPY*-DHFR fusion is markedly slowed and is accompanied by DHFR release in the ER lumen. Thus, folded lumenal domains can impede protein retrotranslocation. The ER lumenal protein Yos9p is required for both release of DHFR and degradation of multiple ERAD substrates. Yos9p forms a complex with substrates and has a sugar binding pocket that is essential for its ERAD function. Nonetheless, substrate recognition persists even when the sugar binding site is mutated or CPY* is unglycosylated. These and other considerations suggest that Yos9p plays a critical role in the bipartite recognition of terminally misfolded glycoproteins.

Amino Acid Sequence↗
PubMed2005

Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins.

Efficiency of interspecies prion transmission decreases as the primary structures of the infectious proteins diverge. Yet, a single prion protein can misfold into multiple infectious conformations, and such differences in "strain conformation" also alter infection specificity. Here, we explored the relationship between prion strains and species barriers by creating distinct synthetic prion forms of the yeast prion protein Sup35. We identified a strain conformation of Sup35 that allows transmission from the S. cerevisiae (Sc) Sup35 to the highly divergent C. albicans (Ca) Sup35 both in vivo and in vitro. Remarkably, cross-species transmission leads to a novel Ca strain that in turn can infect the Sc protein. Structural studies reveal strain-specific conformational differences in regions of the prion domain that are involved in intermolecular contacts. Our findings support a model whereby strain conformation is the critical determinant of cross-species prion transmission while primary structure affects transmission specificity by altering the spectrum of preferred amyloid conformations.

Amyloid↗