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May 1998 - January 2003
February 2010 - present
May 2003 - January 2010
Publications
Publications (61)
Two isoforms of the nuclear pore complex (NPC) have been identified in the yeast S. cerevisiae, which coexist at the periphery of the nucleus and differ by the presence or absence of a nuclear basket. Here, we present a protocol to isolate the two types of NPCs from the same cell extract and dissect their interactomes. We describe steps for powder...
To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRN...
The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; ∼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success r...
The pre-conceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of Assisted Reproductive Technologies (ART; ~25%), additives and adjuvants, such as glucocorticoids, are utilized to improve the succ...
To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRN...
Intron removal from pre-mRNAs is a critical step in the processing of RNA polymerase II transcripts, required to create translation competent mRNAs. In humans, introns account for large portions of the pre-mRNA, with intronic sequences representing about 95% of most pre-mRNA. Intron length varies considerably; introns can be as short as a few to hu...
The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co‐transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore i...
Significance
Alphaviruses are mosquito-borne RNA viruses that cause rash, arthritis, and neurologic disease. Despite a continued risk of outbreaks, there are no licensed interventions for any alphavirus. For progress in control, an understanding of the molecular targets that affect virus replication and virulence is essential. This paper characteri...
mRNAs and lncRNAs assemble with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPsRibonucleoproteins (RNPs)). The assembly of RNPsRibonucleoproteins (RNPs) initiates co-transcriptionally, and their composition and organization is thought to change during the different steps of an RNP life cycle. Modulation of RNP structural organ...
Cells are complex assemblies of molecules organized into organelles and membraneless compartments, each playing important roles in ensuring cellular homeostasis. The different steps of the gene expression pathway take place within these various cellular compartments, and studying gene regulation and RNA metabolism requires incorporating the spatial...
Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of int...
The book provides an overview on the different aspects of gene regulation from an mRNA centric viewpoint, including how mRNA is assembled and self-assembles in a complex consisting of RNA and proteins, and how its ability to be translated at the right time and space depends on many processes acting on the mRNAs, leading to a properly folded complex...
mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single-molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compa...
Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribo...
Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-strande...
Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms...
mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compa...
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome...
We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories....
Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH)...
The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionall...
Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we...
Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single...
After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cel...
Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional pro...
Significance
Our study examines an important aspect of adaptive immunity, namely, the process of effector T-cell activation, which leads to the enhanced expression of lineage-specific cytokine genes upon T-cell receptor (TCR) re-engagement. We found that the TNF locus undergoes TCR-induced homologous allelic pairing, which correlates with biallelic...
Regulating gene expression is a major task for all cellular systems. RNA production and degradation plays a critical role in this process and accurately measuring cellular mRNA levels is essential to understanding gene expression regulation. Classical biochemical assays that study gene expression rely on extracting RNAs from large populations of ce...
Many Saccharomyces cerevisiae genes encode antisense transcripts, some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense (AS) RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single-molecule resolution fluorescent in situ hybridi...
The rate of cell-cycle progression must be tuned in response to nutrient levels to ensure that sufficient materials are synthesized to generate viable daughters. We report that accumulation of the yeast M phase B-cyclin CLB2 mRNA depends on assembly and activation of the heterogeneous nuclear RNA-binding protein (hnRNP) arginine methyltransferase H...
Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure...
The evolutionary 'decision' to store genetic information away from the place of protein synthesis, in a separate compartment, has forced eukaryotic cells to establish a system to transport mRNAs from the nucleus to the cytoplasm for translation. To ensure export to be fast and efficient, cells have evolved a complex molecular interplay that is tigh...
Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluo...
Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur i...
Expression of an individual gene can vary considerably among genetically identical cells because of stochastic fluctuations in transcription. However, proteins comprising essential complexes or pathways have similar abundances and lower variability. It is not known whether coordination in the expression of subunits of essential complexes occurs at...
As the product of transcription and the blueprint for translation, mRNA is the main intermediate product of the gene expression pathway. The ability to accurately determine mRNA levels is, therefore, a major requirement when studying gene expression. mRNA is also a target of different regulatory steps, occurring in different subcellular compartment...
The biogenesis of a localization-competent mRNP begins in the nucleus. It is thought that the coordinated action of nuclear and cytoplasmic components of the localization machinery is required for the efficient export and subsequent subcellular localization of these mRNAs in the cytoplasm. Using quantitative poly(A)(+) and transcript-specific fluor...
Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in...
Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3' end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5' end maturation. Here, we identify R...
Analyzing the expression of single genes in single cells appears minimalistic in comparison to gene expression studies based on more global approaches. However, stimulated by advances in imaging technologies, single-cell studies have become an essential tool in understanding the rules that govern gene expression. This quantitative view of single-ce...
The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more...
Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional act...
INTRODUCTION
The MS2 system provides optimal sensitivity for single-molecule detection in cells. It requires two genetically encoded moieties: a reporter mRNA that contains MS2 binding site (MBS) stem loops and a fluorescent MS2 coat protein (MCP-xFP) that binds to the stem loops with high affinity, thus tagging the mRNA within the cell. This proto...
INTRODUCTION
This protocol describes the application of the MS2 system to the yeast Saccharomyces cerevisiae. ASH1 mRNA tagged with six MS2 repeats (6MBSs) is used to follow the localization of the ASH1 mRNA particles to the bud tip of a haploid yeast cell. W303 yeast cells transformed with pG14-MS2-GFP and pGAL-lacZ-MS2-ASH1 are grown on select me...
INTRODUCTION
This protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a speci...
INTRODUCTION
This protocol describes the use of ImageJ software (freely available from NIH) to analyze particle dynamics in a cell using time-lapse movie frames or image stacks of fluorescent mRNA particles. Maximum intensity projections and kymographs are produced.
INTRODUCTION
The most common way for a cell to respond to internal and external signals is to change its gene expression pattern. This requires the synchronization of regulatory steps along the expression pathway. Biological imaging techniques can be used to visualize and measure such processes in individual live cells in real time. This article di...
Autoregulatory loops often provide precise control of the level of expression of specific genes that encode key regulatory proteins. Here we have defined a pathway by which Yra1p, a yeast mRNA export factor, controls its own expression. We show that YRA1 exon 1 sequences in cis and Yra1p in trans inhibit YRA1 pre-mRNA splicing and commit the pre-mR...
Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3'-untranslated region of the beta-act...
The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appe...
Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells. Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation. Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA...
In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. T...
Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching. ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have bee...
Yra1p/REF participates in mRNA export by recruiting the export receptor Mex67p to messenger ribonucleoprotein (mRNP) complexes.
Yra1p also binds Sub2p, a DEAD box ATPase/RNA helicase implicated in splicing and required for mRNA export. We identified
genetic and physical interactions between Yra1p, Sub2p, and Hpr1p, a protein involved in transcripti...
In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3' processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II...
Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins)
family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling
mRNP export receptor Mex67p in vitro. Here we describe a second nonessentialSaccharomyces cerevisia...
Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic...
We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein
(HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly,
all mutations from the positive selection are located in the N- and C-terminal regions flanking a...
Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors...
