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Gloria A. Brar Publications

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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.

 
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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.  

 
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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. 

 
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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.

 
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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.

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

Ingolia NTBrar 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.

 
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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.

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

Miller, M.P.*, Ünal, E.*Brar, GAAmon, 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. 

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

Ingolia NTBrar 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.

 
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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. 

 
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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.

 
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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.

 
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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.

 
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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.

 
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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.

 
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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.

 
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