Skip to main content

Optimizing In Vitro Pre-mRNA 3′ Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract

  • Protocol
  • First Online:
Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1507))

  • 4206 Accesses

Abstract

Eukaryotic RNA processing steps during mRNA maturation present the cell with opportunities for gene expression regulation. One such step is the pre-mRNA 3′ cleavage reaction, which defines the downstream end of the 3′ untranslated region and, in nearly all mRNA, prepares the message for addition of the poly(A) tail. The in vitro reconstitution of 3′ cleavage provides an experimental means to investigate the roles of the various multi-subunit cleavage factors. Anion-exchange chromatography is the simplest procedure for separating the core mammalian cleavage factors. Here we describe a method for optimizing the in vitro reconstitution of 3′ cleavage activity from the DEAE-sepharose separated HeLa cleavage factors and show how to ensure, or avoid, dependence on creatine phosphate. Important reaction components needed for optimal processing are discussed. We also provide an optimized procedure for preparing small-scale HeLa nuclear extracts from adherent cells for use in 3′ cleavage in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Albig W, Doenecke D (1997) The human histone gene cluster at the D6S105 locus. Hum Genet 101(3):284–294

    Article  CAS  PubMed  Google Scholar 

  2. Conway L, Wickens M (1987) Analysis of mRNA 3′ end formation by modification interference: the only modifications which prevent processing lie in AAUAAA and the poly(A) site. EMBO J 6(13):4177–4184

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Gilmartin GM, McDevitt MA, Nevins JR (1988) Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev 2(5):578–587

    Article  CAS  PubMed  Google Scholar 

  4. Moore CL, Sharp PA (1985) Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell 41(3):845–855

    Article  CAS  PubMed  Google Scholar 

  5. Sperry AO, Berget SM (1986) In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence. Mol Cell Biol 6(12):4734–4741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhang F, Cole CN (1987) Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA. Mol Cell Biol 7(9):3277–3286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Christofori G, Keller W (1988) 3′ cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell 54(6):875–889

    Article  CAS  PubMed  Google Scholar 

  8. Gilmartin GM, Nevins JR (1989) An ordered pathway of assembly of components required for polyadenylation site recognition and processing. Genes Dev 3(12B):2180–2190

    Article  CAS  PubMed  Google Scholar 

  9. McLauchlan J, Moore CL, Simpson S, Clements JB (1988) Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA. Nucleic Acids Res 16(12):5323–5344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Takagaki Y, Ryner LC, Manley JL (1988) Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell 52(5):731–742

    Article  CAS  PubMed  Google Scholar 

  11. Bienroth S, Wahle E, Suter-Crazzolara C, Keller W (1991) Purification of the cleavage and polyadenylation factor involved in the 3′-processing of messenger RNA precursors. J Biol Chem 266(29):19768–19776

    CAS  PubMed  Google Scholar 

  12. Murthy KG, Manley JL (1992) Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem 267(21):14804–14811

    CAS  PubMed  Google Scholar 

  13. Takagaki Y, Manley JL, MacDonald CC, Wilusz J, Shenk T (1990) A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev 4(12A):2112–2120

    Article  CAS  PubMed  Google Scholar 

  14. Ruegsegger U, Beyer K, Keller W (1996) Purification and characterization of human cleavage factor Im involved in the 3′ end processing of messenger RNA precursors. J Biol Chem 271(11):6107–6113

    Article  CAS  PubMed  Google Scholar 

  15. Ruegsegger U, Blank D, Keller W (1998) Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. Mol Cell 1(2):243–253

    Article  CAS  PubMed  Google Scholar 

  16. de Vries H, Ruegsegger U, Hubner W, Friedlein A, Langen H, Keller W (2000) Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J 19(21):5895–5904

    Article  PubMed  PubMed Central  Google Scholar 

  17. Christofori G, Keller W (1989) Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. Mol Cell Biol 9(1):193–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hirose Y, Manley JL (1997) Creatine phosphate, not ATP, is required for 3′ end cleavage of mammalian pre-mRNA in vitro. J Biol Chem 272(47):29636–29642

    Article  CAS  PubMed  Google Scholar 

  19. Takagaki Y, Ryner LC, Manley JL (1989) Four factors are required for 3′-end cleavage of pre-mRNAs. Genes Dev 3(11):1711–1724

    Article  CAS  PubMed  Google Scholar 

  20. Shi Y, Di Giammartino DC, Taylor D, Sarkeshik A, Rice WJ, Yates JR 3rd, Frank J, Manley JL (2009) Molecular architecture of the human pre-mRNA 3′ processing complex. Mol Cell 33(3):365–376. doi:10.1016/j.molcel.2008.12.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ryan K (2007) Pre-mRNA 3′ cleavage is reversibly inhibited in vitro by cleavage factor dephosphorylation. RNA Biol 4(1):26–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cevher MA, Zhang X, Fernandez S, Kim S, Baquero J, Nilsson P, Lee S, Virtanen A, Kleiman FE (2010) Nuclear deadenylation/polyadenylation factors regulate 3′ processing in response to DNA damage. EMBO J 29(10):1674–1687. doi:10.1038/emboj.2010.59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Di Giammartino DC, Li W, Ogami K, Yashinskie JJ, Hoque M, Tian B, Manley JL (2014) RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3′ UTRs. Genes Dev 28(20):2248–2260. doi:10.1101/gad.245787.114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Di Giammartino DC, Shi Y, Manley JL (2013) PARP1 represses PAP and inhibits polyadenylation during heat shock. Mol Cell 49(1):7–17. doi:10.1016/j.molcel.2012.11.005

    Article  CAS  PubMed  Google Scholar 

  25. Kleiman FE, Manley JL (2001) The BARD1-CstF-50 interaction links mRNA 3′ end formation to DNA damage and tumor suppression. Cell 104(5):743–753

    Article  CAS  PubMed  Google Scholar 

  26. Kleiman FE, Wu-Baer F, Fonseca D, Kaneko S, Baer R, Manley JL (2005) BRCA1/BARD1 inhibition of mRNA 3′ processing involves targeted degradation of RNA polymerase II. Genes Dev 19(10):1227–1237. doi:10.1101/gad.1309505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Nazeer FI, Devany E, Mohammed S, Fonseca D, Akukwe B, Taveras C, Kleiman FE (2011) p53 inhibits mRNA 3[prime] processing through its interaction with the CstF/BARD1 complex. Oncogene 30(27):3073–3083

    Article  CAS  PubMed  Google Scholar 

  28. Coqueret O, Gascan H (2000) Functional interaction of STAT3 transcription factor with the cell cycle inhibitor p21WAF1/CIP1/SDI1. J Biol Chem 275(25):18794–18800. doi:10.1074/jbc.M001601200

    Article  CAS  PubMed  Google Scholar 

  29. Folco EG, Lei H, Hsu JL, Reed R (2012) Small-scale nuclear extracts for functional assays of gene-expression machineries. J Vis Exp (64). doi:10.3791/4140

    Google Scholar 

  30. Lee KA, Bindereif A, Green MR (1988) A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech 5(2):22–31

    Article  CAS  PubMed  Google Scholar 

  31. Liu X, Fagotto F (2011) A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci Signal 4(203):pl2. doi:10.1126/scisignal.2002373

    Article  CAS  PubMed  Google Scholar 

  32. Schreiber E, Matthias P, Muller MM, Schaffner W (1989) Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17(15):6419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chabot B (1994) Synthesis and purification of RNA substrates. In: Higgins SJ, Hames BD (eds) RNA processing, a practical approach, vol 1. Oxford University Press, Oxford, pp 1–30

    Google Scholar 

  34. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5):1475–1489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  36. Gilmartin GM (1997) In vitro analysis of mammalian cell mRNA 3′ processing. In: Richter JD (ed) MRNA formation and function. Academic, New York, pp 79–98

    Chapter  Google Scholar 

  37. Moore CL (1990) Preparation of mammalian extracts active in polyadenylation. Methods Enzymol 181:49–74

    Article  CAS  PubMed  Google Scholar 

  38. Wahle E, Keller W (1994) 3′ end-processing of mRNA. In: Higgins SJ, Hames BD (eds) RNA processing: a practical approach, vol 2. Oxford University Press, Oxford UK, pp 1–33

    Google Scholar 

  39. Bentley DL (2014) Coupling mRNA processing with transcription in time and space. Nat Rev Genet 15(3):163–175. doi:10.1038/nrg3662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bai Y, Auperin TC, Chou CY, Chang GG, Manley JL, Tong L (2007) Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Mol Cell 25(6):863–875. doi:10.1016/j.molcel.2007.01.034

    Article  CAS  PubMed  Google Scholar 

  41. Yang Q, Coseno M, Gilmartin GM, Doublie S (2011) Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping. Structure 19(3):368–377. doi:10.1016/j.str.2010.12.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hirose Y, Manley JL (1998) RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395(6697):93–96

    Article  CAS  PubMed  Google Scholar 

  43. Ryan K, Khleborodova A, Pan J, Ryan XP (2009) Small molecule activators of pre-mRNA 3′ cleavage. RNA 15(3):483–492. doi:10.1261/rna.1262509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ryan K, Murthy KG, Kaneko S, Manley JL (2002) Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3′ cleavage. Mol Cell Biol 22(6):1684–1692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wahle E (1991) Purification and characterization of a mammalian polyadenylate polymerase involved in the 3′ end processing of messenger RNA precursors. J Biol Chem 266(5):3131–3139

    CAS  PubMed  Google Scholar 

  46. Khleborodova A, Pan X, Nagre NN, Ryan K. 2016. An investigation into the role of ATP in the mammalian pre-mRNA 3’ cleavage reaction. Biochimie 125:213–222.

    Google Scholar 

Download references

Acknowledgments

This work was supported by grant 5SC1GM083754 to K.R. from the National Institutes of Health. Additional infrastructural support at the City College of New York was provided by the NIH National Center for Research Resources (2G12RR03060-26A1) and the National Institute on Minority Health and Health Disparities (8G12MD007603-27).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kevin Ryan .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Na, M., Valente, S.T., Ryan, K. (2017). Optimizing In Vitro Pre-mRNA 3′ Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract. In: Wajapeyee, N., Gupta, R. (eds) Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation. Methods in Molecular Biology, vol 1507. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6518-2_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6518-2_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6516-8

  • Online ISBN: 978-1-4939-6518-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics