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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Albig W, Doenecke D (1997) The human histone gene cluster at the D6S105 locus. Hum Genet 101(3):284–294
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
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
Moore CL, Sharp PA (1985) Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell 41(3):845–855
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
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
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
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
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
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
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
Murthy KG, Manley JL (1992) Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem 267(21):14804–14811
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
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
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
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
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
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
Takagaki Y, Ryner LC, Manley JL (1989) Four factors are required for 3′-end cleavage of pre-mRNAs. Genes Dev 3(11):1711–1724
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
Ryan K (2007) Pre-mRNA 3′ cleavage is reversibly inhibited in vitro by cleavage factor dephosphorylation. RNA Biol 4(1):26–33
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
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
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
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
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
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
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
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
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
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
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
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
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
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
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
Moore CL (1990) Preparation of mammalian extracts active in polyadenylation. Methods Enzymol 181:49–74
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
Bentley DL (2014) Coupling mRNA processing with transcription in time and space. Nat Rev Genet 15(3):163–175. doi:10.1038/nrg3662
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
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
Hirose Y, Manley JL (1998) RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395(6697):93–96
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
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
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
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.
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
Corresponding author
Editor information
Editors and Affiliations
Rights 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