@article {46736, title = {Cotranscriptional effect of a premature termination codon revealed by live-cell imaging.}, journal = {RNA}, volume = {17}, year = {2011}, month = {2011 Dec}, pages = {2094-107}, abstract = {

Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.

}, keywords = {Cell Line, Codon, Nonsense, Cytoplasm, Gene Expression Regulation, Gene Order, Gene Silencing, Humans, Immunoglobulin mu-Chains, In Situ Hybridization, Fluorescence, Nonsense Mediated mRNA Decay, RNA Splicing, RNA, Messenger, Telomerase, Trans-Activators, Transcription, Genetic}, issn = {1469-9001}, doi = {10.1261/rna.02918111}, author = {de Turris, Valeria and Nicholson, Pamela and Orozco, Rodolfo Zamudio and Singer, Robert H and M{\"u}hlemann, Oliver} } @article {6601, title = {A transgenic mouse for in vivo detection of endogenous labeled mRNA.}, journal = {Nature Methods}, volume = {8}, year = {2011}, month = {2011 Feb}, pages = {165-70}, abstract = {

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3{\textquoteright} untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

}, keywords = {Animals, Cell Survival, Cells, Cultured, Fluorescence, Genetic, In Situ Hybridization, Messenger, Mice, RNA, Transcription, Transgenic}, issn = {1548-7105}, doi = {10.1038/nmeth.1551}, author = {Lionnet, Timoth{\'e}e and Czaplinski, Kevin and Darzacq, Xavier and Shav-Tal, Yaron and Wells, Amber L and Chao, Jeffrey A and Park, Hye Yoon and de Turris, Valeria and Lopez-Jones, Melissa and Singer, Robert H} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

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 complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} }