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.
Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.
Shaked
S
Afik
Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.
Osnat
O
Bartok
Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.|Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
Maxim N
MN
Artyomov
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Alexander A
AA
Shishkin
Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
Sabah
S
Kadri
Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.
Mor
M
Hanan
Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Xiaopeng
X
Zhu
Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Manuel
M
Garber
Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.
Sebastian
S
Kadener
D024509:3' Flanking Region; D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D017344:Genes, Insect; D058977:Molecular Sequence Annotation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
D029721:Drosophila Proteins; D012333:RNA, Messenger; D005092:Exonucleases; D016914:Ribonuclease H