Abstract
The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3′ end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes. Here we present a detailed protocol with minor modifications, which could be broadly applied to investigate transcripts from budding yeast to mammalian organisms.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Hughes TA (2006) Regulation of gene expression by alternative untranslated regions. Trends Genet 22:119–122
Rojas-Duran MF, Gilbert WV (2012) Alternative transcription start site selection leads to large differences in translation activity in yeast. RNA 18:2299–2305
Zhang Z, Dietrich FS (2005) Mapping of transcription start sites in Saccharomyces cerevisiae using 5′ SAGE. Nuc Acids Res 33:2838–2851
Ozsolak F Kapranov P, Foissac S et al (2010) Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation. Cell 143:1018–1029
Miura F, Kawaguchi N, Sese J et al (2006) A large-scale full-length cDNA analysis to explore the budding yeast transcriptome. Proc Natl Acad Sci USA 103:17846–17851
Pelechano V, Wei W, Steinmetz LM (2013) Extensive transcriptional heterogeneity revealed by isoform profiling. Nature 497:127–131
Ng P, Wei CL, Sung WK et al (2005) Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation. Nat Methods 2:105–111
ENCODE Project Consortium, Birney E, Stamatoyannopoulos JA et al (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447:799–816
Zhao XD, Han X, Chew JL et al (2007) Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell Stem Cell 1:286–298
Kang YN, Lai DP, Ooi HS et al (2015) Genome-wide profiling of untranslated regions by paired-end ditag sequencing reveals unexpected transcriptome complexity in yeast. Mol Genet Genomics 290:217–224
Ng P, Wei CL, Ruan Y et al (2007) Paired-end diTagging for transcriptome and genome analysis. Curr Protoc Mol Biol 79:21.12.1–21.12.42
Ni T, Corcoran DL, Rach EA et al (2010) A paired-end sequencing strategy to map the complex landscape of transcription initiation. Nat Methods 7:521–527
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25
Bailey TL, Boden M, Buske FA et al (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31671299) and Natural Science Foundation of Shanghai (19ZR1476100).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kang, Y., Ooi, H.S., Zhao, X. (2019). Transcript Profiling Analysis Through Paired-End Ditag (PET) Approach Coupled with Deep Sequencing Reveals Transcriptome Complexity in Yeast. In: Oliver, S.G., Castrillo, J.I. (eds) Yeast Systems Biology. Methods in Molecular Biology, vol 2049. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9736-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9736-7_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9735-0
Online ISBN: 978-1-4939-9736-7
eBook Packages: Springer Protocols