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Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Abstract

Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms1. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction2,3,4. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5′ end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1–PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1–PAS axis limits pervasive transcription throughout the genome.

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Figure 1: Promoter-proximal PAS-dependent termination of uaRNA.
Figure 2: Asymmetric distribution of PAS and U1 signals flanking coding-gene TSS.
Figure 3: Promoter-proximal cleavage sites are altered upon functional U1 inhibition.
Figure 4: Evolutionary gain and loss of U1 and PAS sites.

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

3′-end sequencing data is deposited in the Gene Expression Omnibus under accession number GSE46433.

References

  1. Djebali, S. et al. Landscape of transcription in human cells. Nature 489, 101–108 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Seila, A. C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Preker, P. et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science 322, 1851–1854 (2008)

    ADS  CAS  Article  PubMed  Google Scholar 

  5. Adelman, K. & Lis, J. T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nature Rev. Genet. 13, 720–731 (2012)

    CAS  Article  PubMed  Google Scholar 

  6. Flynn, R. A., Almada, A. E., Zamudio, J. R. & Sharp, P. A. Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome. Proc. Natl Acad. Sci. USA 108, 10460–10465 (2011)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Preker, P. et al. PROMoter uPstream Transcripts share characteristics with mRNAs and are produced upstream of all three major types of mammalian promoters. Nucleic Acids Res. 39, 7179–7193 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Beaudoing, E., Freier, S., Wyatt, J. R., Claverie, J. M. & Gautheret, D. Patterns of variant polyadenylation signal usage in human genes. Genome Res. 10, 1001–1010 (2000)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 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)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Gil, A. & Proudfoot, N. J. Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit β-globin mRNA 3′ end formation. Cell 49, 399–406 (1987)

    CAS  Article  PubMed  Google Scholar 

  14. MacDonald, C. C., Wilusz, J. & Shenk, T. The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location. Mol. Cell. Biol. 14, 6647–6654 (1994)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Martin, G., Gruber, A. R., Keller, W. & Zavolan, M. Genome-wide analysis of pre-mRNA 3′ end processing reveals a decisive role of human cleavage factor I in the regulation of 3′ UTR length. Cell Rep. 1, 753–763 (2012)

    CAS  Article  PubMed  Google Scholar 

  16. LaCava, J. et al. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713–724 (2005)

    CAS  Article  PubMed  Google Scholar 

  17. Wyers, F. et al. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121, 725–737 (2005)

    CAS  Article  PubMed  Google Scholar 

  18. Vaňáčová, S. et al. A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol. 3, e189 (2005)

    Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Kaida, D. et al. U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664–668 (2010)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Andersen, P. K., Lykke-Andersen, S. & Jensen, T. H. Promoter-proximal polyadenylation sites reduce transcription activity. Genes Dev. 26, 2169–2179 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Zhang, Y. E., Vibranovski, M. D., Landback, P., Marais, G. A. & Long, M. Chromosomal redistribution of male-biased genes in mammalian evolution with two bursts of gene gain on the X chromosome. PLoS Biol. 8, e1000494 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  23. Xie, C. et al. Hominoid-specific de novo protein-coding genes originating from long non-coding RNAs. PLoS Genet. 8, e1002942 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 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)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Connelly, S. & Manley, J. L. A. CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal. Cell 57, 561–571 (1989)

    CAS  Article  PubMed  Google Scholar 

  26. Arigo, J. T., Eyler, D. E., Carroll, K. L. & Corden, J. L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. Mol. Cell 23, 841–851 (2006)

    CAS  Article  PubMed  Google Scholar 

  27. Zhang, L., Ding, Q., Wang, P. & Wang, Z. An upstream promoter element blocks the reverse transcription of the mouse insulin-degrading enzyme gene. Biochem. Biophys. Res. Commun. 430, 26–31 (2013)

    CAS  Article  PubMed  Google Scholar 

  28. Min, I. M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Sigova, A. A. et al. Divergent transcription of lncRNA/mRNA gene pairs in embryonic stem cells. Proc. Natl Acad. Sci. USA 110, 2876–2881 (2013)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011)

    Article  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  32. Grant, C. E., Bailey, T. L. & Noble, W. S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Yeo, G. & Burge, C. B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004)

    CAS  Article  PubMed  Google Scholar 

  34. Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008)

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors acknowledge the service to the MIT community of the late Sean Collier. We would like to thank N. Spies for generously sharing his optimized 3′-end sequencing protocol, C. Lin for providing computational assistance, M. Lindstrom for assistance on constructing Supplementary Figure 11, and S. Chen, A. Chiu, M. Jangi, Q. Liu, J. Wilusz and J. Zamudio for reading of the manuscript. We also thank the Core Facility in the Swanson Biotechnology Center at the David H. Koch Institute for Integrative Cancer Research at MIT for their assistance with high-throughput sequencing. This work was supported by United States Public Health Service grants RO1-GM34277 and R01-CA133404 from the National Institutes of Health (P.A.S.), partially by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute, and by a Public Health Service research grant (GM-085319) from the National Institute of General Medical Sciences (C.B.B.). X.W. is a Howard Hughes Medical Institute International Student Research fellow.

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A.E.A., X.W. and P.A.S. conceived and designed the research. A.E.A. performed experiments. X.W. and A.J.K. performed computational analysis. A.E.A., X.W., C.B.B. and P.A.S. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Phillip A. Sharp.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-11 and Supplementary Tables 1 and 4. (PDF 5591 kb)

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Almada, A., Wu, X., Kriz, A. et al. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499, 360–363 (2013). https://doi.org/10.1038/nature12349

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