Project description:<p>RNA-Seq is an effective method to study the transcriptome, but specialized methods are required to identify 5' ends of transcripts. Several published strategies exist for this specific purpose, but their relative merits have not been systematically analyzed. Here, we directly compare the performance of six such methods - testing five with cellular RNA as well as a novel spike-in RNA assay that helps address interpretation challenges that arise from uncertainties in annotation or RNA processing. Using a single human RNA sample, we constructed and sequenced 18 libraries with these methods and one standard, control RNA-Seq library. We find that the CAGE method performed best for mRNA and that most of its unannotated peaks are supported by evidence from other genomic methods. We then applied CAGE to eight brain-related samples and revealed sample-specific transcription start site (TSS) usage as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.</p>
Project description:Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of the plant BAF-like complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of expressed genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5´-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcriptional start site (TSS) usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and upstream TSS usage suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and alternative TSS usage.
Project description:Role of alternative transcription start sites in muscle growth and development remains largely undetermined. Our WTSS-seq (whole transcriptome start site sequencing) approach to capture 5'-end of RNAs clearly revealed alternative transcription start events responsible for increased muscle mass in AMPK α2 knockout mice.
Project description:Transcription Start Site analysis in Mouse Ter119+ erythroid cells Strand Specific Paired end NanoCage analysis of Total RNA from Mouse Ter119+ erythroid cells