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Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing

Nature Methods volume 10, pages 133–139 (2013)Cite this article

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Abstract

Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3′ region extraction and deep sequencing (3′READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3′ ends revealed over 5,000 previously overlooked pAs (∼8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3′READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3′ end–processing activity in development and differentiation.

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Figure 1: Mapping pAs by 3′READS.
Figure 2: Mouse pAs identified by 3′READS.
Figure 3: Comparison of pAs flanked by A-rich or non–A-rich sequences.
Figure 4: APA of mouse mRNA and lncRNA genes.
Figure 5: Transcript lengthening in cell differentiation and embryonic development.

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Gene Expression Omnibus

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Acknowledgements

We thank Y. Zhao and A. Antes for technical assistance, V. Jin for help with 3T3-L1 differentiation, C. MacDonald (Texas Tech University) for the anti-CstF64 antibody, L. Ford (Bioo Scientific) for pALL-A15 and pALL-A60 plasmids, and S. Gunderson for helpful discussions. This work was funded by the US National Institutes of Health grants (GM084089 and DK094207) to B.T.

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  1. Mainul Hoque and Zhe Ji: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, Newark, New Jersey, USA

    Mainul Hoque, Zhe Ji, Dinghai Zheng, Wenting Luo, Wencheng Li, Bei You, Ji Yeon Park & Bin Tian

  2. Graduate School of Biomedical Sciences, UMDNJ, Newark, New Jersey, USA

    Zhe Ji, Wenting Luo & Bin Tian

  3. Transgenic Core Facility, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA

    Ghassan Yehia

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Contributions

M.H., Z.J. and B.T. conceived of and designed the experiments. M.H., D.Z., W. Luo and B.Y. performed the experiments. Z.J., W. Li and J.Y.P. analyzed the data. G.Y. contributed reagents and materials. M.H., Z.J. and B.T. wrote the paper.

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Correspondence to Bin Tian.

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B.T., M.H., Z.J. and W. Luo are named on the pending US patent application no. PCT/US2012/052122 based on this work.

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Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Discussion (PDF 1087 kb)

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Hoque, M., Ji, Z., Zheng, D. et al. Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing. Nat Methods 10, 133–139 (2013). https://doi.org/10.1038/nmeth.2288

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