Biology subjects

Ingolia, N.

Publications and source records attributed to Ingolia, N..

15 recordsLinked to original sources

bioRxiv2024

Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae

The integrated stress response (ISR) is a conserved eukaryotic signaling pathway that responds to diverse stress stimuli to restore proteostasis. The strength and speed of ISR activation must be tuned properly to allow protein synthesis while maintaining proteostasis. Here, we describe how genetic perturbations change the dynamics of the ISR in budding yeast. We treated ISR dynamics, comprising timecourses of ISR activity across different levels of stress, as a holistic phenotype. We profiled changes in ISR dynamics across thousands of genetic perturbations in parallel using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). We treated cells with sulfometuron methyl, a titratable inhibitor of branched-amino acid synthesis, and measured expression of an ISR reporter. Perturbations to translation such as depletion of aminoacyl-tRNA synthetases or tRNA biogenesis factors reduced cell growth and caused a strikingly proportionate activation of the ISR activation. In contrast, impaired ribosome biogenesis reduced basal ISR activity and weakened ISR dynamics. Reduced ribosome capacity may lower the demand for amino acids and thereby explain these changes. Our work illustrates how CiBER-seq enables high-throughput measurements of complex and dynamic phenotypes that shed light on adaptive and homeostatic mechanisms.

genomics↗

bioRxiv2024

Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing

Sequencing RNAs that are biologically processed or degraded to less than [~]100 nucleotides typically involves multi-step, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced Ordered Two-Template Relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced non-productive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 picograms (less than 1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or post-transcriptional modifications.

biochemistry↗

bioRxiv2024

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.

molecular biology↗

bioRxiv2024

CRISPRi with barcoded expression reporters dissects regulatory networks in human cells

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-{kappa}B) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.

genomics↗

bioRxiv2024

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

genomics↗

bioRxiv2024

Dbp1 is a low performance paralog of RNA helicase Ded1 that drives impaired translation and heat stress response

Ded1 and Dbp1 are paralogous conserved RNA helicases that enable translation initiation in yeast. Ded1 has been heavily studied but the role of Dbp1 is poorly understood. We find that the expression of these two helicases is controlled in an inverse and condition-specific manner. In meiosis and other long-term starvation states, Dbp1 expression is upregulated and Ded1 is downregulated, whereas in mitotic cells, Dbp1 expression is extremely low. Inserting the DBP1 ORF in place of the DED1 ORF cannot replace the function of Ded1 in supporting translation, partly due to inefficient mitotic translation of the DBP1 mRNA, dependent on features of its ORF sequence but independent of codon optimality. Global measurements of translation rates and 5 leader translation, activity of mRNA-tethered helicases, ribosome association, and low temperature growth assays show that--even at matched protein levels--Ded1 is more effective than Dbp1 at activating translation, especially for mRNAs with structured 5 leaders. Ded1 supports halting of translation and cell growth in response to heat stress, but Dbp1 lacks this function, as well. These functional differences in the ability to efficiently mediate translation activation and braking can be ascribed to the divergent, disordered N- and C-terminal regions of these two helicases. Altogether, our data show that Dbp1 is a "low performance" version of Ded1 that cells employ in place of Ded1 under long-term conditions of nutrient deficiency.

genomics↗

bioRxiv2023

eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3 untranslated region (3-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

molecular biology↗

bioRxiv2022

Ribosome stalling during selenoprotein translation exposes a ferroptosis vulnerability in cancer

Ferroptosis is a regulated, iron-dependent form of necrosis that is triggered by the accumulation of oxidatively damaged phospholipids1-3. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting phospholipid hydroperoxides into non-toxic lipid alcohols4, 5. Ferroptosis has been implicated in the pathology of several degenerative conditions and inhibiting GPX4 activity has emerged as a therapeutic strategy to induce cancer cell death1, 2. However, many cancer cell lines are resistant to GPX4 inhibition6, and the mechanisms that regulate GPX4 activity and ferroptosis resistance remain incompletely understood. Here, employing a synthetic lethal CRISPR-Cas9 screen in a triple negative breast cancer (TNBC) cell line, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor. LRP8 is upregulated in cancer, and we find that it promotes ferroptosis resistance in cancer cells in both 2-dimensional (2-D) cell culture and 3-dimensional (3-D) spheroid models. Mechanistically, loss of LRP8 decreases cellular selenium levels, resulting in the reduced expression of a subset of selenoproteins, including GPX4. Remarkably, the reduction in GPX4 is not due to the classic hierarchical selenoprotein regulatory program7, 8. Instead, our findings demonstrate that the translation of GPX4 is severely impaired in the selenium-deficient LRP8 knockout (KO) cells due to extensive ribosome stalling at the inefficiently decoded GPX4 selenocysteine (SEC) UGA codon, which results in ribosome collisions and early translation termination. Thus, our findings reveal ribosome stalling and collisions during GPX4 translation as targetable ferroptosis vulnerabilities in cancer cells.

cancer biology↗

bioRxiv2021

Surveying the global landscape of post-transcriptional regulators

Numerous proteins regulate gene expression by modulating mRNA translation and decay. In order to uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze [~]50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.

molecular biology↗

bioRxiv2021

The N-terminal and central domains of CoV-2 nsp1 play key functional roles in suppression of cellular gene expression and preservation of viral gene expression

Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.

molecular biology↗

bioRxiv2020

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

eIF2 phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

molecular biology↗

bioRxiv2020

CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

To realize the promise of CRISPR/Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We address this challenge by profiling transcriptional, translational, and post-translational reporters using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach connects an entire library of guides to their individual phenotypic consequences using pooled sequencing. We show that CiBER-seq profiling fully recapitulates the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged tRNA accumulation activated ISR reporter transcription. Surprisingly, tRNA insufficiency also activated the reporter, independent of the Gcn2 kinase that senses uncharged tRNAs. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

genomics↗

bioRxiv2020

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding interpretable loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, but no genome-scale guide libraries have been reported. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

genomics↗

bioRxiv2018

An Accessible Continuous-Culture Turbidostat for Pooled Analysis of Complex Libraries

We present an accessible, robust continuous-culture turbidostat system that enables biologists to generate and phenotypically analyze highly complex libraries in yeast and bacteria. Our system has many applications in genomics and systems biology; here, we demonstrate three of these uses. We first measure how the growth rate of budding yeast responds to limiting nitrogen at steady state and in a dynamically varying environment. We also demonstrate the direct selection of a diverse, genome-scale protein fusion library in liquid culture. Finally, we perform a comprehensive mutational analysis of the essential gene RPL28 in budding yeast, mapping sequence constraints on its wild-type function and delineating the binding site of the drug cycloheximide through resistance mutations. Our system can be constructed and operated with no specialized skills or equipment and applied to study genome-wide mutant pools and diverse libraries of sequence variants under well-defined growth conditions.

systems biology↗

bioRxiv2018

Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

Diverse ribonucleoprotein complexes control messenger RNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-Seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS) provides new insights into the organization of translation initiation complexes on active mRNAs, as well as exposing unanticipated complexity in stress granule composition, and provides a powerful and general approach to explore the spatial environment of macromolecules.

genomics↗