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

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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References

  1. Colgan, D.F. & Manley, J.L. Mechanism and regulation of mRNA polyadenylation. Genes Dev. 11, 2755–2766 (1997).

    Article  CAS  Google Scholar 

  2. Proudfoot, N.J. Ending the message: poly(A) signals then and now. Genes Dev. 25, 1770–1782 (2011).

    Article  CAS  Google Scholar 

  3. Tian, B. & Graber, J.H. Signals for pre-mRNA cleavage and polyadenylation. Wiley Interdiscip. Rev. RNA 3, 385–396 (2011).

    Article  Google Scholar 

  4. Shi, Y. et al. Molecular architecture of the human pre-mRNA 3′ processing complex. Mol. Cell 33, 365–376 (2009).

    Article  CAS  Google Scholar 

  5. Tian, B., Hu, J., Zhang, H. & Lutz, C.S. A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res. 33, 201–212 (2005).

    Article  CAS  Google Scholar 

  6. Di Giammartino, D.C., Nishida, K. & Manley, J.L. Mechanisms and consequences of alternative polyadenylation. Mol. Cell 43, 853–866 (2011).

    Article  CAS  Google Scholar 

  7. Lutz, C.S. & Moreira, A. Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression. Wiley Interdiscip. Rev. RNA 2, 23–31 (2011).

    Article  Google Scholar 

  8. Zhang, H., Lee, J.Y. & Tian, B. Biased alternative polyadenylation in human tissues. Genome Biol. 6, R100 (2005).

    Article  Google Scholar 

  9. Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

  10. Ji, Z., Lee, J.Y., Pan, Z., Jiang, B. & Tian, B. Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc. Natl. Acad. Sci. USA 106, 7028–7033 (2009).

    Article  CAS  Google Scholar 

  11. Sandberg, R., Neilson, J.R., Sarma, A., Sharp, P.A. & Burge, C.B. Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science 320, 1643–1647 (2008).

    Article  CAS  Google Scholar 

  12. Mayr, C. & Bartel, D.P. Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138, 673–684 (2009).

    Article  CAS  Google Scholar 

  13. Singh, P. et al. Global changes in processing of mRNA 3′ untranslated regions characterize clinically distinct cancer subtypes. Cancer Res. 69, 9422–9430 (2009).

    Article  CAS  Google Scholar 

  14. Flavell, S.W. et al. Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection. Neuron 60, 1022–1038 (2008).

    Article  CAS  Google Scholar 

  15. Chen, L.L. & Carmichael, G.G. Long noncoding RNAs in mammalian cells: what, where, and why? Wiley Interdiscip. Rev. RNA 1, 2–21 (2010).

    Article  CAS  Google Scholar 

  16. Wang, K.C. & Chang, H.Y. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904–914 (2011).

    Article  CAS  Google Scholar 

  17. Lee, J.Y., Yeh, I., Park, J.Y. & Tian, B. PolyA_DB 2: mRNA polyadenylation sites in vertebrate genes. Nucleic Acids Res. 35, D165–D168 (2007).

    Article  CAS  Google Scholar 

  18. Brockman, J.M. et al. PACdb: PolyA Cleavage Site and 3′-UTR Database. Bioinformatics 21, 3691–3693 (2005).

    Article  CAS  Google Scholar 

  19. Nam, D.K. et al. Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription. Proc. Natl. Acad. Sci. USA 99, 6152–6156 (2002).

    Article  CAS  Google Scholar 

  20. Schmidt, M.J. & Norbury, C.J. Polyadenylation and beyond: emerging roles for noncanonical poly(A) polymerases. Wiley Interdiscip. Rev. RNA 1, 142–151 (2010).

    Article  CAS  Google Scholar 

  21. Wlotzka, W., Kudla, G., Granneman, S. & Tollervey, D. The nuclear RNA polymerase II surveillance system targets polymerase III transcripts. EMBO J. 30, 1790–1803 (2011).

    Article  CAS  Google Scholar 

  22. Derti, A. et al. A quantitative atlas of polyadenylation in five mammals. Genome Res. 22, 1173–1183 (2012).

    Article  CAS  Google Scholar 

  23. Shepard, P.J. et al. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17, 761–772 (2011).

    Article  CAS  Google Scholar 

  24. Jan, C.H., Friedman, R.C., Ruby, J.G. & Bartel, D.P. Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469, 97–101 (2011).

    Article  CAS  Google Scholar 

  25. ENCODE Project Consortium. A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 9, e1001046 (2011).

  26. Lee, J.Y., Park, J.Y. & Tian, B. Identification of mRNA polyadenylation sites in genomes using cDNA sequences, expressed sequence tags, and Trace. Methods Mol. Biol. 419, 23–37 (2008).

    Article  CAS  Google Scholar 

  27. Tian, B., Pan, Z. & Lee, J.Y. Widespread mRNA polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing. Genome Res. 17, 156–165 (2007).

    Article  CAS  Google Scholar 

  28. Ozsolak, F. et al. Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation. Cell 143, 1018–1029 (2010).

    Article  CAS  Google Scholar 

  29. Mangone, M. et al. The landscape of C. elegans 3′ UTRs. Science 329, 432–435 (2010).

    Article  CAS  Google Scholar 

  30. Fu, Y. et al. Differential genome-wide profiling of tandem 3′ UTRs among human breast cancer and normal cells by high-throughput sequencing. Genome Res. 21, 741–747 (2011).

    Article  CAS  Google Scholar 

  31. Fox-Walsh, K., Davis-Turak, J., Zhou, Y., Li, H. & Fu, X.D. A multiplex RNA-seq strategy to profile poly(A+) RNA: application to analysis of transcription response and 3′ end formation. Genomics 98, 266–271 (2011).

    Article  CAS  Google Scholar 

  32. Yoon, O.K. & Brem, R.B. Noncanonical transcript forms in yeast and their regulation during environmental stress. RNA 16, 1256–1267 (2010).

    Article  CAS  Google Scholar 

  33. Ji, Z. et al. Transcriptional activity regulates alternative cleavage and polyadenylation. Mol. Syst. Biol. 7, 534 (2011).

    Article  Google Scholar 

  34. Ji, Z. & Tian, B. Reprogramming of 3′ untranslated regions of mRNAs by alternative polyadenylation in generation of pluripotent stem cells from different cell types. PLoS ONE 4, e8419 (2009).

    Article  Google Scholar 

  35. Berg, M.G. et al. U1 snRNP determines mRNA length and regulates isoform expression. Cell 150, 53–64 (2012).

    Article  CAS  Google Scholar 

  36. Zhang, Y. et al. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc. Natl. Acad. Sci. USA 106, 19860–19865 (2009).

    Article  CAS  Google Scholar 

  37. Wang, Z., Tollervey, J., Briese, M., Turner, D. & Ule, J. CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo. Methods 48, 287–293 (2009).

    Article  Google Scholar 

  38. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  39. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).

    Article  CAS  Google Scholar 

  40. Hu, J., Lutz, C.S., Wilusz, J. & Tian, B. Bioinformatic identification of candidate cis-regulatory elements involved in human mRNA polyadenylation. RNA 11, 1485–1493 (2005).

    Article  CAS  Google Scholar 

  41. Zhang, C. & Darnell, R.B. Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat. Biotechnol. 29, 607–614 (2011).

    Article  CAS  Google Scholar 

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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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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.

Corresponding author

Correspondence to Bin Tian.

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Competing interests

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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