Raymond Chen

All Publications

• Cell-type specific features of circular RNA expression. PLoS genetics Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., Brown, P. O. 2013; 9 (9)

  Abstract

  Thousands of loci in the human and mouse genomes give rise to circular RNA transcripts; at many of these loci, the predominant RNA isoform is a circle. Using an improved computational approach for circular RNA identification, we found widespread circular RNA expression in Drosophila melanogaster and estimate that in humans, circular RNA may account for 1% as many molecules as poly(A) RNA. Analysis of data from the ENCODE consortium revealed that the repertoire of genes expressing circular RNA, the ratio of circular to linear transcripts for each gene, and even the pattern of splice isoforms of circular RNAs from each gene were cell-type specific. These results suggest that biogenesis of circular RNA is an integral, conserved, and regulated feature of the gene expression program.

  View details for DOI 10.1371/journal.pgen.1003777

  View details for PubMedID 24039610

  View details for PubMedCentralID PMC3764148

• Dynamic Localization of Fus3 Mitogen-Activated Protein Kinase Is Necessary To Evoke Appropriate Responses and Avoid Cytotoxic Effects MOLECULAR AND CELLULAR BIOLOGY Chen, R. E., Patterson, J. C., Goupil, L. S., Thorner, J. 2010; 30 (17): 4293-4307

  Abstract

  Cellular responses to many external stimuli are mediated by mitogen-activated protein kinases (MAPKs). We investigated whether dynamic intracellular movement contributes to the spatial and temporal characteristics of the responses elicited by a prototypic MAPK, Fus3, in the mating pheromone response pathway in budding yeast (Saccharomyces cerevisiae). Confining Fus3 in the nucleus, via fusion to a histone H2B, reduced MAPK activation and diminished all responses (pheromone-induced gene expression, cell cycle arrest, projection formation, and mating). Elimination of MAPK phosphatases restored more robust outputs for all responses, indicating that nuclear sequestration impedes full MAPK activation but does not abrogate its functional competence. Restricting Fus3 to the plasma membrane, via fusion to a lipid-modified CCaaX motif, led to MAPK hyperactivation yet severely impaired all response outputs. Fus3-CCaaX also caused aberrant cell morphology and a proliferation defect. Unlike similar phenotypes induced by pathway hyperactivation via upstream components, these deleterious effects were independent of the downstream transcription factor Ste12. Thus, appropriate cellular responses require free subcellular MAPK transit to disseminate MAPK activity optimally because preventing dynamic MAPK movement either markedly impaired signal-dependent activation and/or resulted in improper biological outputs.

  View details for DOI 10.1128/MCB.00361-10

  View details for Web of Science ID 000280762900017

  View details for PubMedID 20584989

• Systematic Epistasis Analysis of the Contributions of Protein Kinase A- and Mitogen-Activated Protein Kinase-Dependent Signaling to Nutrient Limitation-Evoked Responses in the Yeast Saccharomyces cerevisiae GENETICS Chen, R. E., Thorner, J. 2010; 185 (3): 855-870

  Abstract

  Cellular responses to environmental stimuli require conserved signal transduction pathways. In budding yeast (Saccharomyces cerevisiae), nutrient limitation induces morphological changes that depend on the protein kinase A (PKA) pathway and the Kss1 mitogen-activated protein kinase (MAPK) pathway. It was unclear to what extent and at what level there is synergy between these two distinct signaling modalities. We took a systematic genetic approach to clarify the relationship between these inputs. We performed comprehensive epistasis analysis of mutants lacking different combinations of all relevant pathway components. We found that these two pathways contribute additively to nutrient limitation-induced haploid invasive growth. Moreover, full derepression of either pathway rendered it individually sufficient for invasive growth and thus, normally, both are required only because neither is maximally active. Furthermore, in haploids, the MAPK pathway contributes more strongly than the PKA pathway to cell elongation and adhesion, whereas nutrient limitation-induced unipolar budding is independent of both pathways. In contrast, in diploids, upon nutrient limitation the MAPK pathway regulates cell elongation, the PKA pathway regulates unipolar budding, and both regulate cell adhesion. Thus, although there are similarities between haploids and diploids, cell type-specific differences clearly alter the balance of the signaling inputs required to elicit the various nutrient limitation-evoked cellular behaviors.

  View details for DOI 10.1534/genetics.110.115808

  View details for Web of Science ID 000281906800013

  View details for PubMedID 20421603

• Stress resistance and signal fidelity independent of nuclear MAPK function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Westfall, P. J., Patterson, J. C., Chen, R. E., Thorner, J. 2008; 105 (34): 12212-12217

  Abstract

  Elevated external solute stimulates a conserved MAPK cascade that elicits responses that maintain osmotic balance. The yeast high-osmolarity glycerol (HOG) pathway activates Hog1 MAPK (mammalian ortholog p38alpha/SAPKalpha), which enters the nucleus and induces expression of >50 genes, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we show here that cells lacking the karyopherin required for Hog1 nuclear import or in which Hog1 is anchored at the plasma membrane (or both) can withstand long-term hyperosmotic challenge by ionic and nonionic solutes without exhibiting the normal change in transcriptional program (comparable with hog1Delta cells), as judged by mRNA hybridization and microarray analysis. For such cells to survive hyperosmotic stress, systematic genetic analysis ruled out the need for any Hog1-dependent transcription factor, the Hog1-activated MAPKAP kinases, or ion, glycerol, and water channels. By contrast, enzymes needed for glycerol production were essential for viability. Thus, control of intracellular glycerol formation by Hog1 is critical for maintenance of osmotic balance but not transcriptional induction of any gene.

  View details for DOI 10.1073/pnas.0805797105

  View details for Web of Science ID 000258905700023

  View details for PubMedID 18719124

• Function and regulation in MAPK signaling pathways: Lessons learned from the yeast Saccharomyces cerevisiae BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Chen, R. E., Thorner, J. 2007; 1773 (8): 1311-1340

  Abstract

  Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells.

  View details for DOI 10.1016/j.bbamcr.2007.05.003

  View details for Web of Science ID 000249086200014

  View details for PubMedID 17604854

• Systems biology approaches in cell signaling research GENOME BIOLOGY Chen, R. E., Thorner, J. 2005; 6 (10)

  Abstract

  The use of methods for global and quantitative analysis of cells is providing new systems-level insights into signal transduction processes. Recent studies reveal important information about the rates of signal transmission and propagation, help establish some general regulatory characteristics of multi-tiered signaling cascades, and illuminate the combinatorial nature of signaling specificity in cell differentiation.

  View details for DOI 10.1186/gb-2005-6-10-235

  View details for Web of Science ID 000232679600004

  View details for PubMedID 16207364

• Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae MOLECULAR BIOLOGY OF THE CELL Versele, M., Gullbrand, B., Shulewitz, M. J., Cid, V. J., Bahmanyar, S., Chen, R. E., Barth, P., Alber, T., Thorner, J. 2004; 15 (10): 4568-4583

  Abstract

  Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

  View details for DOI 10.1091/mbc.E04-04-0330

  View details for Web of Science ID 000224162200020

  View details for PubMedID 15282341

• Characterization of Nkx3.2 DNA binding specificity and its requirement for somitic chondrogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Kim, D. W., Kempf, H., Chen, R. E., Lassar, A. B. 2003; 278 (30): 27532-27539

  Abstract

  We have previously shown that Nkx3.2, a member of the NK class of homeoproteins, functions as a transcriptional repressor to promote somitic chondrogenesis. However, it has not been addressed whether Nkx3.2 can bind to DNA in a sequence-specific manner and whether DNA binding by Nkx3.2 is required for its biological activity. In this work, we employed a DNA binding site selection assay, which identified TAAGTG as a high affinity Nkx3.2 binding sequence. Sequence-specific binding of Nkx3.2 to the TAAGTG motif in vitro was confirmed by electrophoretic mobility shift assays, and mutagenesis of this sequence revealed that HRAGTG (where H represents A, C, or T, and R represents A or G) comprises the consensus DNA binding site for Nkx3.2. Consistent with these findings, the expression of a reporter gene containing reiterated Nkx3.2 binding sites was repressed in vivo by Nkx3.2 co-expression. In addition, we have generated a DNA nonbinding point mutant of Nkx3.2 (Nkx3.2-N200Q), which contains an asparagine to glutamine missense mutation in the homeodomain. Interestingly, despite being defective in DNA binding, Nkx3.2-N200Q still retains its intrinsic transcriptional repressor function. Finally, we demonstrate that unlike wild-type Nkx3.2, Nkx3.2-N200Q is unable to activate the chondrocyte differentiation program in somitic mesoderm, indicating that DNA binding by Nkx3.2 is critical for this factor to induce somitic chondrogenesis.

  View details for DOI 10.1074/jbc.M301461200

  View details for Web of Science ID 000184242700026

  View details for PubMedID 12746429