Gloria A. Brar Publications
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.
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.
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.
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.
Beyond the Triplet Code: Context Cues Transform Translation. Cell (2016).
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.
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.
Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes. Cell Reports (2014).
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.
A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern. Genes and Development (2013).
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).
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).
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).
Here, we detail the protocol for ribosome profiling, a method for identifying coding regions and quantifying new protein synthesis, globally and in vivo.
High-Resolution View of the Yeast Meiotic Program Revealed by Ribosome Profiling. Science (2012).
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).
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).
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).
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.
The multiple roles of cohesin in meiotic chromosome morphogenesis and pairing. Molecular Biology of the Cell (2009)
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.
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.
Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis. Nature (2006).
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.
The FK506 binding protein Fpr3 counteracts protein phosphatase 1 to maintain meiotic recombination checkpoint activity. Cell (2005)
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.