ABSTRACT
We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a, and Dcp2 and the termination factor TTF2 coimmunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease "torpedo" that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2, and TTF2 localize near transcription start sites (TSSs) by ChIP-seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors Xrn2 and TTF2 shifted polymerase away from the TSS toward upstream and downstream distal positions. This redistribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the "torpedo" mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated cotranscriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.
Subject(s)
Exoribonucleases/physiology , RNA Polymerase II/physiology , RNA Stability , RNA, Messenger/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , HEK293 Cells , HeLa Cells , Humans , Models, Genetic , Protein Interaction Mapping , Transcription Factors/metabolism , Transcription Factors/physiology , Transcription, GeneticABSTRACT
Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.
Subject(s)
Extracellular Matrix Proteins/chemistry , Proteome/analysis , Solutions/chemistry , Ultrasonics , Animals , Cell Culture Techniques , Cells, Cultured , Chromatography, Liquid/methods , Epithelial Cells/cytology , Epithelial Cells/metabolism , Extracellular Matrix Proteins/metabolism , Female , Mammary Glands, Animal/chemistry , Mammary Glands, Animal/cytology , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methodsABSTRACT
Bimetallic complexes based on the binucleating ligand N,N,N',N'-tetrakis[(2-benzimidazolyl)methyl]-2-hydroxy-1,3-diaminopropane (1L) and its new toluoyl ester derivative (2L) catalyze the hydrolysis of phosphorus triesters at ambient temperature with activities rivalling the fastest known systems.
