An efficient method for genome-wide polyadenylation site mapping and RNA quantification

Genome Biology Unit, European Molecular Biology Laboratory and Genomics Core Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 01/2013; 41(5). DOI: 10.1093/nar/gks1249
Source: PubMed


The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability,
subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific
manner. This method, termed 3′T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to
start directly after the poly(A) tail into the 3′-untranslated regions (UTR). Our comparative analysis demonstrates that it
outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced
from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe
that overlap of convergent 3′-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression
of the yeast genome.

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Available from: Stefan Wilkening
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    • "In this case, at least 600 additional overlapping coding genes have been identified (Sanna et al., 2008). We wished to address the question of the fate of 3 0 -overlapping messengers in the model organism Saccharomyces cerevisiae , where hundreds of 3 0 -overlapping mRNA result from convergent gene transcription and can theoretically form mRNA duplexes (Pelechano et al., 2013; Wilkening et al., 2013). Convergent gene expression has already been shown to lead to transcriptional interference in S. cerevisiae. "
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    ABSTRACT: Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.
    Full-text · Article · Sep 2015 · Cell Reports
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    • "Such approaches provide inexpensive and relatively simple tools to monitor eukaryotic gene expression . Moreover, the recent realization that condition-dependent alternative 3 ′ -UTR cleavage and polyadenylation is common in eukaryotes, and can radically alter mRNA metabolism (Sandberg et al. 2008; Mayr and Bartel 2009; Di Giammartino et al. 2011), has led to further approaches to identify the frequency and position of alternative mRNA ends (Beck et al. 2010; Mangone et al. 2010; Ozsolak et al. 2010; Yoon and Brem 2010; Fu et al. 2011; Jan et al. 2011; Shepard et al. 2011; Ulitsky et al. 2012; Wilkening et al. 2013). The poly(A) tail is more than just a convenient purification hook however; polyadenylation of protein-coding RNA is essential for eukaryotic life and normal protein translation. "
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    ABSTRACT: A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide. © 2015 Harrison et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    Full-text · Article · Jun 2015 · RNA
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    • "Another method which avoids sequencing through the poly(A) tail is described by Wilkening et al. [69]. In this method, named 3′T-fill, the poly(A) stretch is filled in with dTTPs before the sequencing reaction starts. "
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    ABSTRACT: Technological advances in the sequencing field support in-depth characterization of the transcriptome. Here, we review genome-wide RNA sequencing methods used to investigate specific aspects of gene expression and its regulation, from transcription to RNA processing and translation. We discuss tag-based methods for studying transcription, alternative initiation and polyadenylation events, shotgun methods for detection of alternative splicing, full-length RNA sequencing for the determination of complete transcript structures, and targeted methods for studying the process of transcription and translation. With the ensemble of technologies available, it is now possible to obtain a comprehensive view on transcriptome complexity and the regulation of transcript diversity.
    Full-text · Article · May 2014 · Cellular and Molecular Life Sciences CMLS
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