Yehuda Brody

In vivo dynamics of RNA polymerase II transcription

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min−1, much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.

The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.

The life of an mRNA in space and time

Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed β-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic β-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the β-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo.

Phosphorylation of Protein Kinase Cδ on Distinct Tyrosine Residues Induces Sustained Activation of Erk1/2 via Down-regulation of MKP-1 ROLE IN THE APOPTOTIC EFFECT OF ETOPOSIDE

The mechanism underlying the important role of protein kinase Cδ (PKCδ) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCδ in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCδ since the phosphorylation of Erk1/2 was inhibited by a PKCδ-KD mutant and PKCδ small interfering RNA. We recently reported that phosphorylation of PKCδ on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCδtyrosine mutants, we found that the phosphorylation of PKCδon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCδ was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCδ. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCδ and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCδon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.

Transcription and splicing: When the twain meet

Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

Visualizing transcription in real-time

The transcriptional process is at the center of the gene expression pathway. In eukaryotes, the transcription of protein-coding genes into messenger RNAs is performed by RNA polymerase II. This enzyme is directed to bind at upstream gene sequences by the aid of transcription factors that assemble transcription-competent complexes. A series of biochemical and structural modifications render the polymerase transcriptionally active so that it can proceed from an initiation state into a functional elongating phase. Recent experimental efforts have attempted to visualize these processes as they take place on genes in living cells and to quantify the kinetics of in vivo transcription.

Quantifying the Ratio of Spliceosome Components Assembled on Pre-mRNA

RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.

GeneRetriever: software to extract all genes and transcripts in between two genetic markers to assist design of human custom microarrays

180 BioTechniques Vol. 39, No. 2 (2005) Identifying the genes that are responsible for human genetic disorders with complex inheritance patterns (e.g., multigenic diseases) has proven to be more difficult than anticipated. Indeed, when the mode of inheritance is unknown, classical parametric linkage studies are not relevant. Nonparametric linkage analysis can help approximate the candidate loci, but this type of analysis often leads to the identification of large intervals (10–20 cM) that may contain hundreds of genes, thus rendering the candidate gene approach rather tedious (1). Adding an expression screening may significantly reduce the number of candidate genes. Microarray expression studies of the genes located within such genetic intervals should be performed on relevant tissues (2). The design of a custom microrarray containing probes of all the genes located in the intervals of interest is required to carry out such experiments. Designing such microarrays involves gathering much additional information concerning these genes, in particular, name, transcript accession numbers, or nucleotide sequences. Collecting these data manually is time-consuming and very error-prone. GeneRetriever is a Perl-based data mining tool developed to automate, accelerate, and secure the process of locally retrieving user-chosen comprehensive information about human genes or transcripts located between two genetic markers. As annotation strategies are specific for each database, we implemented a database parameter entry that allows collection of data from either the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih. gov) or Ensembl (www.ensembl.org) databases (3,4). Then, several options make it possible to define which data should be included in the returned gene/transcript table. These options are clustered into three parts: (i) genespecific data; (ii) transcript-specific data; and (iii) expression analysis data. Gene-specific options include database (either NCBI or Ensembl) identifier, Hugo Gene Nomenclature Committee symbol (5), gene description, DNA strand (plus or minus), type of gene (known or predicted), cytogenetic localization, summary of functional annotations, Entrez Gene identifier (6), web address of either NCBI or Ensembl gene page, and the number of transcript variants. Additional transcript information is optionally available, such as Ensembl transcript or RefSeq accession numbers. Structural data, including transcript size, number of exons, and size of the longest exon, can also be added to the query. These data may be useful when working with predicted genes; for instance, the relevance of predicted genes composed of one single exon of <100 bp can be questioned. Nucleotide sequences can also be gathered; in addition, the retrieved size of the 3′ end of transcript sequences can be userdefined in order to facilitate the design of transcript-specific oligonucleotides for microarray spotting. Since GeneRetriever was developed to help the design of a custom gene expression study, the list of genes can be directly linked either to the available BENCHMARKS

Measuring transcription dynamics in living cells using a photobleaching approach

The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics.