2024

Truncated protein isoforms generate diversity of protein localization and function in yeast

Cell Systems (2024)

Andrea L. Higdon, Nathan H. Won, Gloria A. Brar

Genome-wide measurement of ribosome occupancy on mRNAs has enabled empirical identification of translated regions, but high-confidence detection of coding regions that overlap annotated coding regions has remained challenging. Here, we report a sensitive and robust algorithm that revealed the translation of 388 N-terminally truncated proteins in budding yeast—more than 30-fold more than previously known. We extensively experimentally validated them and defined two classes. The first class lacks large portions of the annotated protein and tends to be produced from a truncated transcript. We show that two such cases, Yap5truncation and Pus1truncation, have condition-specific regulation and distinct functions from their respective annotated isoforms. The second class of truncated protein isoforms lacks only a small region of the annotated protein and is less likely to be produced from an alternative transcript isoform. Many display different subcellular localizations than their annotated counterpart, representing a common strategy for dual localization of otherwise functionally identical proteins.

Dbp1, a low-performance RNA helicase paralogous to Ded1, makes cells insensitive to heat stress despite stress granule formation

Biorxiv (2024)

Emily N Powers, Naohiro Kuwayama, Matej Siketanc, Camila Sousa, Kendra Reynaud, Marko Jovanovic, Maria Hondele, Nicholas T Ingolia, Gloria A Brar

*April update: in review, version 2.0 with new and exciting data coming soon to biorxiv

Ded1 and Dbp1 are paralogous and conserved RNA helicases with divergent N- and C-termini, and function in enabling translation initiation in yeast. Ded1 has been heavily studied but the role of Dbp1 is poorly understood. 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 DBP1 in place of DED1 cannot replace the function of Ded1 in supporting translation, partly due to inefficient translation of the DBP1 ORF itself in mitosis. Global measurements of translation, activity of mRNA-tethered helicases, and 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 housekeeping gene translation and cell growth in response to heat stress, and Dbp1 is also deficient at this function. Surprisingly, the inability of Dbp1 to quell cell growth in response to heat is not based on a deficiency in stress granule formation, suggesting that formation of these cellular condensates does not mediate Ded1 function in the heat stress response. Altogether, our data show that Dbp1 is a “low performance” and stress-insensitive version of Ded1 that cells employ in place of Ded1 under long-term conditions of nutrient deficiency.

2023

Fix it, don't trash it: Ribosome maintenance by chaperone-mediated repair of damaged subunits

Molecular Cell (2023)

Silvan Spiri & Gloria Ann Brar

Acute stressors or normal cellular function may result in ribosomal protein damage, which threatens the functional ribosome pool and translation. We describe the work of Yang et al., who show that chaperones can extract damaged ribosomal proteins and replace them with newly synthesized versions to repair mature ribosomes.

Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression

JCB (2023)

Helen Vander Wende, Mounika Gopi, Megan Onyundo, Claudia Medrano, Temiloluwa Adanlawo, Gloria Ann Brar

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case—Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients—is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1’s role in human disease.

2022

Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation

eLIFE (2022)

Emily Nicole Powers, Charlene Chan, Ella Doron-Mandel,Lidia Llacsahuanga Allcca, Jenny Kim Kim, Marko Jovanovic,Gloria Ann Brar

Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1. This behavior resembled the ‘neighboring gene effect’ (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.

Gametogenesis: Exploring an Endogenous Rejuvenation Program to Understand Cellular Aging and Quality Control

Annual Reviews Genetics (2022)

Tina L. Sing, Gloria A. Brar, and Elçin Ünal

Gametogenesis is a conserved developmental program whereby a diploid progenitor cell differentiates into haploid gametes, the precursors for sexually reproducing organisms. In addition to ploidy reduction and extensive organelle remodeling, gametogenesis naturally rejuvenates the ensuing gametes, leading to resetting of life span. Excitingly, ectopic expression of the gametogenesis-specific transcription factor Ndt80 is sufficient to extend life span in mitotically dividing budding yeast, suggesting that meiotic rejuvenation pathways can be repurposed outside of their natural context. In this review, we highlight recent studies of gametogenesis that provide emerging insight into natural quality control, organelle remodeling, and rejuvenation strategies that exist within a cell. These include selective inheritance, programmed degradation, and de novo synthesis, all of which are governed by the meiotic gene expression program entailing many forms of noncanonical gene regulation. Finally, we highlight critical questions that remain in the field and provide perspective on the implications of gametogenesis research on human health span.

The promises and pitfalls of specialized ribosomes

Molecular Cell (2022)

Maria Barna, Katrin Karbstein, David Tollervey, Davide Ruggero, Gloria Brar, Eric Lieberman Greer, Jonathan D. Dinman

The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.

Faculty Profile: Gloria Brar

Berkeley Science Review (2022)

Héctor L. Torres Vera

Meiotic cDNA libraries reveal gene truncations and mitochondrial proteins important for competitive fitness in Saccharomyces cerevisiae

Genetics (2022)

Tina L Sing, Katie Conlon, Stephanie H Lu, Nicole Madrazo, Kaitlin Morse, Juliet C Barker, Ina Hollerer, Gloria A Brar, Peter H Sudmant , Elçin Ünal

Gametogenesis is an evolutionarily conserved developmental program whereby a diploid progenitor cell undergoes meiosis and cellular remodeling to differentiate into haploid gametes, the precursors for sexual reproduction. Even in the simple eukaryotic organism Saccharomyces cerevisiae, the meiotic transcriptome is very rich and complex, thereby necessitating new tools for functional studies. Here, we report the construction of 5 stage-specific, inducible complementary DNA libraries from meiotic cells that represent over 84% of the genes found in the budding yeast genome. We employed computational strategies to detect endogenous meiotic transcript isoforms as well as library-specific gene truncations. Furthermore, we developed a robust screening pipeline to test the effect of each complementary DNA on competitive fitness. Our multiday proof-of-principle time course revealed 877 complementary DNAs that were detrimental for competitive fitness when overexpressed. The list included mitochondrial proteins that cause dose-dependent disruption of cellular respiration as well as library-specific gene truncations that expose a dominant negative effect on competitive growth. Together, these high-quality complementary DNA libraries provide an important tool for systematically identifying meiotic genes, transcript isoforms, and protein domains that are important for a specific biological function.

Developmentally regulated selective autophagy determines ER inheritance by gametes.

Autophagy (2022)

George M. Otto and Gloria A. Brar

The endoplasmic reticulum (ER) carries out essential cellular functions ranging from protein trafficking to metabolite signaling. ER function is maintained in part by quality control pathways including ER degradation by selective autophagy (reticulophagy) during conditions of cellular stress. Reticulophagy is known to be important for cellular responses to starvation and protein folding stress, but no natural role during development had been identified. While investigating ER remodeling during the conserved cell differentiation process of meiosis in budding yeast, we unexpectedly observed developmentally regulated reticulophagy that was driven by expression of the autophagy receptor Atg40. This reticulophagy was coordinated with massive morphological rearrangement of the ER, including movement of most cortical ER away from the cell periphery. As meiotic reticulophagy prevents specific ER subpopulations from being inherited by gametes, we propose that it serves a quality control role, preventing deleterious material from being passed on to subsequent generations.

2021

The power of perpetual collaboration: An interview with Elcin Ünal and Gloria Brar

Molecular Cell (2021)

Here, Elcin Ünal and Gloria Brar tell us how the Br-Ün Lab came to be, the cons, but mostly the pros, of running a joint lab, as well as their philosophies in research and mentoring a diverse group of scientists.

Programmed cortical ER collapse drives selective ER degradation and inheritance in yeast meiosis

JCB (2021)

Otto GM, Cheunkarndee T, Leslie JM, Brar GA

The endoplasmic reticulum (ER) carries out essential and conserved cellular functions, which depend on the maintenance of its structure and subcellular distribution. Here, we report developmentally regulated changes in morphology and composition of the ER during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. A subset of the cortical ER collapses away from the plasma membrane at anaphase II, thus separating into a spatially distinct compartment. This programmed collapse depends on the transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER-plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which relies on ER collapse and is regulated by timed expression of the autophagy receptor Atg40. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.

Performing Ribosome Profiling to Assess Translation in Vegetative and Meiotic Yeast Cells

Methods in Molecular Biology. (2021)

Powers EN & Brar GA

Ribosome profiling, first developed in 2009, is the gold standard for quantifying and qualifying changes to translation genome-wide (Ingolia et al., Science, 2009). Though first designed and optimized in vegetative budding yeast, it has since been modified and specialized for use in diverse cellular states in yeast, as well as in bacteria, plants, human cells, and many other organisms (Ingolia et al. Science, 2009, reviewed in (Ingolia et al., Cold Spring Harb Perspect Biol, 2019; Brar and Weissman, Nat Rev Mol Cell Biol, 2015)). Here we report the current ribosome profiling protocol used in our lab to study genome-wide changes to translation in budding yeast undergoing the developmental process of meiosis (Brar et al., Science, 2012; Cheng et al., Cell, 2018). We describe this protocol in detail, including the following steps: collection and flash freezing samples, cell lysis and extract preparation, sucrose gradient centrifugation and monosome collection, RNA extraction, library preparation, and library quality control. Almost every step presented here should be directly applicable to performing ribosome profiling in other eukaryotic cell types or cell states.

Rules are made to be broken: a "simple" model organism reveals the complexity of gene regulation

Current Genetics. (2021)

Higdon AL & Brar GA

Global methods for assaying translation have greatly improved our understanding of the protein-coding capacity of the genome. In particular, it is now possible to perform genome-wide and condition-specific identification of translation initiation sites through modified ribosome profiling methods that selectively capture initiating ribosomes. Here we discuss our recent study applying such an approach to meiotic and mitotic timepoints in the simple eukaryote, budding yeast, as an example of the surprising diversity of protein products-many of which are non-canonical-that can be revealed by such methods. We also highlight several key challenges in studying non-canonical protein isoforms that have precluded their prior systematic discovery. A growing body of work supports expanded use of empirical protein-coding region identification, which can help relieve some of the limitations and biases inherent to traditional genome annotation approaches. Our study also argues for the adoption of less static views of gene identity and a broader framework for considering the translational capacity of the genome.

2020

Global mapping of translation initiation sites by TIS profiling in budding yeast

STAR Protocols (2020)

Hollerer I, Powers EN, Brar GA

Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs in vivo by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells.

Angelika Amon

Developmental Cell (2020)

Brar GA and Ünal E

This is a commissioned tribute for our mentor, Angelika Amon. She was a truly exceptional scientist and human being. We attempted to capture her vibrant, hilarious, and generous spirit, as well as her unusual, wonderful approach to science and mentorship.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast. Cell Systems (2020)

Eisenberg AR, Higdon AL, Hollerer I, Fields AP, Jungreis I, Diamond PD, Kellis M, Jovanovic M, Brar GA.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster. G3 (2020)

Jorgensen V, Chen J, Vander Wende H, Harris DE, McCarthy A, Breznak S, Wong-Deyrup SW, Chen Y, Rangan P, Brar GA, Sawyer EM, Chan LY, Ünal E.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

2019

Global translation inhibition yields condition-dependent de-repression of ribosome biogenesis mRNAs. Nucleic Acids Research (2019)

Cheng Z and Brar GA

Ribosome biogenesis (RiBi) is an extremely energy intensive process that is critical for gene expres- sion. It is thus highly regulated, including through the tightly coordinated expression of over 200 RiBi genes by positive and negative transcriptional regu- lators. We investigated RiBi regulation as cells initi- ated meiosis in budding yeast and noted early tran- scriptional activation of RiBi genes, followed by their apparent translational repression 1 hour (h) after stimulation to enter meiosis. Surprisingly, in the representative genes examined, measured translational repression depended on their promoters rather than mRNA regions. Further investigation revealed that the signature of this regulation in our data depended on pre-treating cells with the translation inhibitor, cycloheximide (CHX). This treatment, at 1 h in meiosis, but not earlier, rapidly resulted in accumulation of RiBi mRNAs that were not translated. This effect was also seen in with CHX pre-treatment of cells grown in media lacking amino acids. For NSR1, this effect de- pended on the –150 to –101 region of the promoter, as well as the RiBi transcriptional repressors Dot6 and Tod6. Condition-specific RiBi mRNA accumulation was also seen with translation inhibitors that are dissimilar from CHX, suggesting that this phenomenon might represent a feedback response to global translation inhibition.

Small and Large Ribosomal Subunit Deficiencies Lead to Distinct Gene Expression Signatures that Reflect Cellular Growth Rate. Molecular Cell (2019)

Cheng Z, Mugler CF, Keskin A, Hodapp S, Chen L L-Y, Weis K, Mertins P, Regev A, Jovanovic M, Brar GA

Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked from or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene subclasses, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.

2018

Precise Post-translational Tuning Occurs for Most Protein Complex Components during Meiosis. Cell (2018)

Eisenberg AR, Higdon A*, Keskin A, Hodapp S, Jovanovic M, Brar GA (*equal contributions)

Protein degradation is known to be a key component of expression regulation for individual genes, but its global impact on gene expression has been difficult to determine. We analyzed a parallel gene expression dataset of yeast meiotic differentiation, identifying instances of coordinated protein-level decreases to identify new cases of regulated meiotic protein degradation, including of ribosomes and targets of the meiosis-specific anaphase-promoting complex adaptor Ama1. Comparison of protein and translation measurements over time also revealed that, although meiotic cells are capable of synthesizing protein complex members at precisely matched levels, they typically do not. Instead, the members of most protein complexes are synthesized imprecisely, but their protein levels are matched, indicating that wild-type eukaryotic cells routinely use post-translational adjustment of protein complex partner levels to achieve proper stoichiometry. Outlier cases, in which specific complex components show divergent protein-level trends, suggest timed regulation of these complexes.

Global Proteome Remodeling during ER Stress Involves Hac1-Driven Expression of Long Undecoded Transcript Isoforms. Developmental Cell (2018)

Van Dalfsen KM, Hodapp S, Keskin A, Otto GM, Berdan CA, Higdon A, Cheunkarndee T, Nomura DK, Jovanovic M, Brar GA

Cellular stress responses often require transcription-based activation of gene expression to promote cellular adaptation. Whether general mechanisms exist for stress-responsive gene downregulation is less clear. A recently defined mechanism enables both up- and downregulation of protein levels for distinct gene sets by the same transcription factor via coordinated induction of canonical mRNAs and long undecoded transcript isoforms (LUTIs). We analyzed parallel gene expression datasets to determine whether this mechanism contributes to the conserved Hac1-driven branch of the unfolded protein response (UPR), indeed observing Hac1-dependent protein downregulation accompanying the upregulation of ER-related proteins that typifies UPR activation. Proteins downregulated by Hac1-driven LUTIs include those with electron transport chain (ETC) function. Abrogated ETC function improves the fitness of UPR-activated cells, suggesting functional importance to this regulation. We conclude that the UPR drives large-scale proteome remodeling, including coordinated up- and downregulation of distinct protein classes, which is partly mediated by Hac1-induced LUTIs.

The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs. Nature (2018)

Guenther U-P, Weinberg DE, Zubradt MM, Tedeschi FA, Stawicki BN, Zagore LL, Brar GA, Licatalosi DD, Bartel DP, Weissman JS & Jankowsky E

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2–4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p–RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5′ untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5′ untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5′ untranslated regions.

Evidence for an Integrated Gene Repression Mechanism based on mRNA Isoform Toggling in Human Cells (equal collaboration with Ünal lab). G3 (2019)

Hollerer I, Barker JC*, Jorgensen V*, Tresenrider A, Dugast-Darzacq C, Darzacq X, Chan LY, Tjian R, Ünal E**, Brar GA**  (*, ** equal contributions)

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2. We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

Seq-ing answers: uncovering the unexpected in global gene regulation. Current Genetics (2018)

Otto GM and Brar GA

The development of techniques for measuring gene expression globally has greatly expanded our understanding of gene regulatory mechanisms in depth and scale. We can now quantify every intermediate and transition in the canonical pathwayof gene expression—from DNA to mRNA to protein—genome-wide. Employing such measurements in parallel can producerich datasets, but extracting the most information requires careful experimental design and analysis. Here, we argue for thevalue of genome-wide studies that measure multiple outputs of gene expression over many timepoints during the course of a natural developmental process. We discuss our findings from a highly parallel gene expression dataset of meiotic differentiation, and those of others, to illustrate how leveraging these features can provide new and surprising insight into fundamental mechanisms of gene regulation.

Pervasive, Coordinated Protein-Level Changes Driven by Transcript Isoform Switching during Meiosis. Cell (2018)

Cheng Z*, Otto GM*, Powers EN, Keskin A, Mertins P, Carr SA, Jovanovic M, Brar GA (*equal contributions)

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 50 extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced.

m6A and eIF2α-ⓟ Team Up to Tackle ATF4 Translation during Stress. Mol Cell (2018)

Powers EN and Brar GA

While m6A modification of mRNAs is now known to be widespread, the cellular roles of this modification remain largely mysterious. In this issue of Molecular Cell, Zhou et al. (2018) show that m6A modification unexpectedly contributes to the established uORF- and eIF2α-ⓟ-dependent mechanism of ATF4 translational regulation in response to stress.

2017

Strategies and Challenges in Identifying Function for Thousands of sORF-Encoded Peptides in Meiosis. Proteomics (2017).

Hollerer I, Higdon A, Brar GA

Recent genomic analyses have revealed pervasive translation from formerly unrecognized short open reading frames (sORFs) during yeast meiosis. Despite their short length, which has caused these regions to be systematically overlooked by traditional gene annotation approaches, meiotic sORFs share many features with classical genes, implying the potential for similar types of cellular functions. We found that sORF expression accounts for approximately 10-20% of the cellular translation capacity in yeast during meiotic differentiation and occurs within well-defined time windows, suggesting the production of relatively abundant peptides with stage-specific meiotic roles from these regions. Here, we provide arguments supporting this hypothesis and discuss sORF similarities and differences, as a group, to traditional protein coding regions, as well as challenges in defining their specific functions.

2016

Beyond the Triplet Code: Context Cues Transform Translation. Cell (2016).

Brar GA

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

2015

Ribosome profiling reveals the what, when, where, and how of protein synthesis. Nature Reviews Molecular and Cell Biology (2015).

Brar GA and Weissman JS. 

Ribosome profiling, the deep sequencing of ribosome-protected mRNA fragments, represents a powerful tool for globally monitoring protein translation in vivo. The method has enabled discovery of the gene expression regulation underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing the first systematic approach for experimental coding region annotation. Here we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including prominently in identifying thousands of novel translated short ORFs and alternative translation products.

2014

Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes. Cell Reports (2014).

 Ingolia NT, Brar GA, Stern-Ginossar N, Harris N,  Talhouarne GJS, Jackson SE, Wills MR, and Weissman JS. 

Ribosome profiling is a in vivo experimental approach for detecting and quantifying translation. Its use in several systems has identified unexpected apparent translated regions, including uORFs and short ORFs on messages previously thought to be noncoding. Here, we validate the translation of these regions in ES cells, Human Cytomegalovirus infected cells, and meiotic budding yeast. We also introduce a metric called FLOSS that allows post hoc analysis of ribosome profiling data to determine the likelihood that unexpected ribosome footprints represent true translation.  

2013

A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern. Genes and Development (2013).

Berchowitz LE, Gajadhar AS, van Werven FJ, De Rosa AA, Samoylova ML, Brar GA, Xu Y, Xiao C, Futcher B, Weissman JS, White FM, Amon A. 

Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I-meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 5' untranslated region of CLB3. At the onset of meiosis II,  a decrease in Rim4 protein levels, alleviates this translational repression. 

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell (2013).

Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Onn Brandman, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. 

The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide RNA. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, positioning CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Aneuploid yeast strains exhibit defects in cell growth and passage through START. Molecular Biology of the Cell (2013).

Thorburn RR, Gonzalez C, Brar GA, Christen S, Carlile TM, Ingolia NT, Sauer U, Weissman JS, Amon A. 

Aneuploidy, a chromosome content that is not a multiple of the haploid karyotype, is associated with reduced fitness in all organisms analyzed to date. In budding yeast aneuploidy causes cell proliferation defects, with many different aneuploid strains exhibiting a delay in G1, a cell cycle stage governed by extracellular cues, growth rate, and cell cycle events. Here we characterize this G1 delay. We show that aneuploid yeast strains exhibit a growth defect during G1. Our results indicate that aneuploidy frequently interferes with the ability of cells to grow and, as with many other cellular stresses, entry into the cell cycle.

Genome-wide annotation and quantitation of translation by ribosome profiling. Current Protocols in Molecular Biology (2013).

Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. 

Here, we detail the protocol for ribosome profiling, a method for identifying coding regions and quantifying new protein synthesis, globally and in vivo.

2012

High-Resolution View of the Yeast Meiotic Program Revealed by Ribosome Profiling. Science (2012).

Brar GA, Yassour M, Friedman N, Regev A, Ingolia NT, Weissman JS. 

Meiosis is a complex developmental process that generates haploid cells from diploid progenitors. We measured mRNA abundance and protein production through the yeast meiotic program and found strong, stage-specific expression for most genes, achieved through control of both mRNA levels and translational efficiency. Monitoring of protein production timing revealed uncharacterized recombination factors and extensive organellar remodeling. Meiotic translation is also shifted toward noncanonical sites, including short open reading frames (ORFs) on unannnotated transcripts and upstream regions of known transcripts (uORFs). Ribosome occupancy at near-cognate uORFs was associated with more efficient ORF translation; by contrast, some AUG uORFs, often exposed by regulated 5' leader extensions, acted competitively. This work reveals pervasive translational control in meiosis and helps to illuminate the molecular basis of the broad restructuring of meiotic cells.

Meiosis I chromosome segregation is established by inhibiting microtubule-kinetochore interactions in Prophase I. eLife (2012).

Miller, M.P.*, Ünal, E.*, Brar, GA, Amon, A. (*equal contributions). 

In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Here we show that preventing microtubule-kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division. Furthermore we find that restricting outer kinetochore assembly through translational regulation of Ndc80 contributes to preventing premature engagement of microtubules with kinetochores. 

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nature Protocols (2012).

Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. 

Here, we outline the protocol for ribosome profiling, a method for identifying coding regions and quantifying new protein synthesis, globally and in vivo.

Proto-genes and de novo gene birth. Nature (2012).

Carvunis AR, Rolland T, Wapinski I, Calderwood MA, Yildirim MA, Simonis N, Charloteaux B, Hidalgo CA, Barbette J, Santhanam B, Brar GA, Weissman JS, Regev A, Thierry-Mieg N, Cusick ME, Vidal M. 

Novel protein-coding genes can arise either through re-organization of pre-existing genes or de novo. Here we formalize an evolutionary model according to which functional genes evolve de novo through transitory proto-genes generated by widespread translational activity in non-genic sequences. Testing this model at the genome scale in Saccharomyces cerevisiae, we detect translation of hundreds of short species-specific open reading frames (ORFs) located in non-genic sequences. These translation events seem to provide adaptive potential. In line with our model, we establish that S. cerevisiae ORFs can be placed within an evolutionary continuum ranging from non-genic sequences to genes. We identify ~1,900 candidate proto-genes among S. cerevisiae ORFs and find that de novo gene birth from such a reservoir may be more prevalent than sporadic gene duplication. 

2009

The multiple roles of cohesin in meiotic chromosome morphogenesis and pairing. Molecular Biology of the Cell (2009)

Brar GA, Hochwagen A, Ee L, Amon A.

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. In addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, we show that cohesin phosphorylation is not only important for its removal, but that cohesin's meiotic prophase functions are distinct. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a central player in multiple meiotic events.

2008

Emerging roles for centromeres in meiosis I chromosome segregation. Nature Reviews Genetics (2008).

Brar GA, Amon A.

Centromeres are an essential and conserved feature of eukaryotic chromosomes, yet recent research indicates that we are just beginning to understand the numerous roles that centromeres have in chromosome segregation. During meiosis I, in particular, centromeres seem to function in many processes in addition to their canonical role in assembling kinetochores, the sites of microtubule attachment. Here we summarize recent advances that place centromeres at the centre of meiosis I, and discuss how these studies affect a variety of basic research fields.

The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing. Proceedings of the National Academy of Sciences (2008).

Nguyen HH, Aronchik I, Brar GA, Nguyen DH, Bjeldanes LF, Firestone GL.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G(1) cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Here, we demonstrate that I3C disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct noncompetitive inhibition of human neutrophil elastase enzymatic activity. Our results y implicate the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.

2006

Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis. Nature (2006).

Brar GA, Kiburz BM, Zhang Y, Kim JE, White F, Amon A. 

During meiosis, cohesins, protein complexes that hold sister chromatids together, are lost from chromosomes in a step-wise manner. Loss of cohesins from chromosome arms is necessary for homologous chromosomes to segregate during meiosis I. Retention of cohesins around centromeres until meiosis II is required for the accurate segregation of sister chromatids. Here we show that phosphorylation of the cohesin subunit Rec8 contributes to step-wise cohesin removal. Our data further implicate two other key regulators of meiotic chromosome segregation, the cohesin protector Sgo1 and meiotic recombination in bringing about the step-wise loss of cohesins and thus the establishment of the meiotic chromosome segregation pattern.

2005

The FK506 binding protein Fpr3 counteracts protein phosphatase 1 to maintain meiotic recombination checkpoint activity. Cell (2005)

Hochwagen A, Tham WH, Brar GA, Amon A.

The meiotic recombination checkpoint delays gamete precursors until DNA breaks created during recombination are repaired and chromosome structure has been restored. Here, we show that the FK506 binding protein Fpr3 prevents premature adaptation to damage and thus serves to maintain recombination checkpoint activity. Impaired checkpoint function is observed both in cells lacking FPR3 and in cells treated with rapamycin, a small molecule inhibitor that binds to the proline isomerase (PPIase) domain of Fpr3. FPR3 functions in the checkpoint through controlling protein phosphatase 1 (PP1).  Our findings define a branch of the recombination checkpoint involved in the adaptation to persistent chromosomal damage.

Indole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms, and subcellular localization of the CDK2 protein complex. Journal of Biological Chemistry (2005).

Garcia HH, Brar GA, Nguyen DH, Bjeldanes LF, Firestone GL.

Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E.