Defining the 5' and 3' landscape of the Drosophila transcriptome with Exo-seq and RNaseH-seq
Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5΄- and 3΄-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.
© 2017 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 25 May 2016; Revision Received: 10 February 2017; Accepted: 15 February 2017; Published: 22 February 2017. We thank Reut Ashwal-Fluss for help with some of the computational analysis and Patrick McDonel for help with protocol optimization. Funding: International Human Frontiers Science Program Organization [PG #31/2011 to S.K.]; European Research Council Consolidator Grant [ERC #647989 to S.K.]; Defense Advanced Research Project Agency [D13AP00074 to M.G., S.K.]; National Institute of Health [U01HG007910-01, UL1TR001453-01, 5U54HD082013-02 to M.G.). Funding for open access charge: European Research Council Consolidator Grant [ERC #647989 to S.K.]. Conflict of interest statement: Alexander A. Shishkin is an inventor on a massively multiplexed RNA-sequencing patent (publication number WO 2014152155 A1). The authors declare that they don't have any other conflict of interest.
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