Abstract
Quiescence (G0) is defined as an assortment of cell cycle arrested states that exhibit distinct properties. Leukemias harbor a subpopulation of G0 cells that can be enriched by growth factor deprivation or serum starvation. Target site reporters with shortened poly(A) tails show translation activation by microRNAs, via a noncanonical mechanism, when introduced into the nucleus of G0 cells. This is because recruitment by the activation causing FXR1a-microRNA-protein complex (FXR1a-microRNP) is nuclear and requires shortened poly(A) tails to avoid repressive factors and canonical translation. When introduced into the cytoplasm, target mRNAs and microRNAs are directed toward repression rather than translation activation. Leukemic cell lines are difficult to transfect but can be routinely nucleofected—where in vitro transcribed mRNA reporters and microRNAs are introduced into the nucleus of G0 leukemic cells. Nucleofection of a microRNA target reporter and either cognate, targeting microRNA, or control microRNA, into the nucleus of G0 cells, enables analysis of translation activation by microRNAs in G0. We discuss a modified protocol that we developed for transfection of mRNAs along with microRNAs to test translation regulation by microRNAs in G0 leukemic cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Pardee AB (1974) A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci U S A 71(4):1286–1290
Aragon AD, Rodriguez AL, Meirelles O, Roy S, Davidson GS, Tapia PH, Allen C, Joe R, Benn D, Werner-Washburne M (2008) Characterization of differentiated quiescent and nonquiescent cells in yeast stationary-phase cultures. Mol Biol Cell 19(3):1271–1280. doi:10.1091/mbc.E07-07-0666
Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4(3):e83. doi:10.1371/journal.pbio.0040083
Ng SW, Mitchell A, Kennedy JA, Chen WC, McLeod J, Ibrahimova N, Arruda A, Popescu A, Gupta V, Schimmer AD, Schuh AC, Yee KW, Bullinger L, Herold T, Gorlich D, Buchner T, Hiddemann W, Berdel WE, Wormann B, Cheok M, Preudhomme C, Dombret H, Metzeler K, Buske C, Lowenberg B, Valk PJ, Zandstra PW, Minden MD, Dick JE, Wang JC (2016) A 17-gene stemness score for rapid determination of risk in acute leukaemia. Nature 540(7633):433–437. doi:10.1038/nature20598
Kreso A, Dick JE (2014) Evolution of the cancer stem cell model. Cell Stem Cell 14(3):275–291. doi:10.1016/j.stem.2014.02.006
Meacham CE, Morrison SJ (2013) Tumour heterogeneity and cancer cell plasticity. Nature 501(7467):328–337. doi:10.1038/nature12624
Crews LA, Jamieson CH (2013) Selective elimination of leukemia stem cells: hitting a moving target. Cancer Lett 338(1):15–22. doi:10.1016/j.canlet.2012.08.006
Bhola PD, Mar BG, Lindsley RC, Ryan JA, Hogdal LJ, Vo TT, DeAngelo DJ, Galinsky I, Ebert BL, Letai A (2016) Functionally identifiable apoptosis-insensitive subpopulations determine chemoresistance in acute myeloid leukemia. J Clin Investig 126(10):3827–3836. doi:10.1172/JCI82908
Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321(5892):1095–1100. doi:10.1126/science.1155998
Tavaluc RT, Hart LS, Dicker DT, El-Deiry WS (2007) Effects of low confluency, serum starvation and hypoxia on the side population of cancer cell lines. Cell Cycle 6(20):2554–2562. doi:10.4161/cc.6.20.4911
Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138(4):645–659. doi:10.1016/j.cell.2009.06.034
Lemons JM, Feng XJ, Bennett BD, Legesse-Miller A, Johnson EL, Raitman I, Pollina EA, Rabitz HA, Rabinowitz JD, Coller HA (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8(10):e1000514. doi:10.1371/journal.pbio.1000514
Lindeman GJ, Visvader JE (2010) Insights into the cell of origin in breast cancer and breast cancer stem cells. Asia Pac J Clin Oncol 6(2):89–97. doi:10.1111/j.1743-7563.2010.01279.x
Salony SX, Alves CP, Dey-Guha I, Ritsma L, Boukhali M, Lee JH, Chowdhury J, Ross KN, Haas W, Vasudevan S, Ramaswamy S (2016) AKT inhibition promotes nonautonomous cancer cell survival. Molecular cancer therapeutics 15(1):142–153. doi:10.1158/1535-7163.MCT-15-0414
Dey-Guha I, Wolfer A, Yeh AC, GA J, Darp R, Leon E, Wulfkuhle J, Petricoin EF 3rd, Wittner BS, Ramaswamy S (2011) Asymmetric cancer cell division regulated by AKT. Proc Natl Acad Sci U S A 108(31):12,845–12,850. doi:10.1073/pnas.1109632108
Zheng X, Seshire A, Ruster B, Bug G, Beissert T, Puccetti E, Hoelzer D, Henschler R, Ruthardt M (2007) Arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha-positive leukemic stem cells. Haematologica 92(3):323–331
Li L, Bhatia R (2011) Stem cell quiescence. Clin Cancer Res: Off J Am Assoc Cancer Res 17(15):4936–4941. doi:10.1158/1078-0432.CCR-10-1499
Barnes DJ, Melo JV (2006) Primitive, quiescent and difficult to kill: the role of non-proliferating stem cells in chronic myeloid leukemia. Cell Cycle 5(24):2862–2866. doi:10.4161/cc.5.24.3573
Goldman J, Gordon M (2006) Why do chronic myelogenous leukemia stem cells survive allogeneic stem cell transplantation or imatinib: does it really matter? Leuk Lymphoma 47(1):1–7. doi:10.1080/10428190500407996
Reed JC (1998) Molecular biology of chronic lymphocytic leukemia: implications for therapy. Semin Hematol 35(3 Suppl 3):3–13
Giles FJ, DeAngelo DJ, Baccarani M, Deininger M, Guilhot F, Hughes T, Mauro M, Radich J, Ottmann O, Cortes J (2008) Optimizing outcomes for patients with advanced disease in chronic myelogenous leukemia. Semin Oncol 35(1 Suppl 1):S1–17.; quiz S18–20. doi:10.1053/j.seminoncol.2007.12.002
Krause A, Luciana M, Krause F, Rego EM (2010) Targeting the acute myeloid leukemia stem cells. Anti Cancer Agents Med Chem 10(2):104–110
Besancon R, Valsesia-Wittmann S, Puisieux A, Caron de Fromentel C, Maguer-Satta V (2009) Cancer stem cells: the emerging challenge of drug targeting. Curr Med Chem 16(4):394–416
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. doi:10.1016/j.cell.2011.02.013
Le Tonqueze O, Kollu S, Lee S, Al-Salah M, Truesdell SS, Vasudevan S (2016) Regulation of monocyte induced cell migration by the RNA binding protein, FXR1. Cell Cycle 15(14):1874–1882. doi:10.1080/15384101.2016.1189040
Sandberg R, Neilson JR, Sarma A, Sharp PA, Burge CB (2008) Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science 320(5883):1643–1647. doi:10.1126/science.1155390
Mayr C, Bartel DP (2009) Widespread shortening of 3’UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138(4):673–684. doi:10.1016/j.cell.2009.06.016
Cheung TH, Rando TA (2013) Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol 14(6):329–340. doi:10.1038/nrm3591
Bukhari SI, Truesdell SS, Lee S, Kollu S, Classon A, Boukhali M, Jain E, Mortensen RD, Yanagiya A, Sadreyev RI, Haas W, Vasudevan S (2016) A Specialized Mechanism of Translation Mediated by FXR1a-Associated MicroRNP in Cellular Quiescence. Mol Cell 61(5):760–773. doi:10.1016/j.molcel.2016.02.013
Lee S, Truesdell SS, Bukhari SI, Lee JH, LeTonqueze O, Vasudevan S (2014) Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages. Proc Natl Acad Sci U S A 111(41):E4315–E4322. doi:10.1073/pnas.1320477111
Loayza-Puch F, Drost J, Rooijers K, Lopes R, Elkon R, Agami R (2013) p53 induces transcriptional and translational programs to suppress cell proliferation and growth. Genome Biol 14(4):R32. doi:10.1186/gb-2013-14-4-r32
Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136(4):731–745. doi:10.1016/j.cell.2009.01.042
Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12(1):21–35. doi:10.1038/nrm3025
Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to activation: microRNAs can up-regulate translation. Science 318(5858):1931–1934. doi:10.1126/science.1149460
Mortensen RD, Serra M, Steitz JA, Vasudevan S (2011) Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNA-protein complexes (microRNPs). Proc Natl Acad Sci U S A 108(20):8281–8286. doi:10.1073/pnas.1105401108
Truesdell SS, Mortensen RD, Seo M, Schroeder JC, Lee JH, LeTonqueze O, Vasudevan S (2012) MicroRNA-mediated mRNA translation activation in quiescent cells and oocytes involves recruitment of a nuclear microRNP. Sci Rep 2:842. doi:10.1038/srep00842
Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16(7):421–433. doi:10.1038/nrg3965
He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7):522–531. doi:10.1038/nrg1379
Ameres SL, Zamore PD (2013) Diversifying microRNA sequence and function. Nat Rev Mol Cell Biol 14(8):475–488. doi:10.1038/nrm3611
Fabian MR, Sundermeier TR, Sonenberg N (2010) Understanding how miRNAs post-transcriptionally regulate gene expression. Prog Mol Subcell Biol 50:1–20. doi:10.1007/978-3-642-03103-8_1
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. doi:10.1016/j.cell.2009.01.002
Bukhari SI, Vasudevan S (2017) FXR1a-associated microRNP: A driver of specialized non-canonical translation in quiescent conditions. RNA Biol 14(2):137–145. doi:10.1080/15476286.2016.1265197
Vasudevan S, Steitz JA (2007) AU-rich-element-mediated upregulation of translation by FXR1 and Argonaute 2. Cell 128(6):1105–1118
Dube M, Huot ME, Khandjian EW (2000) Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast. BMC Genet 1:4. doi:10.1186/1471-2156-1-4
Siomi MC, Zhang Y, Siomi H, Dreyfuss G (1996) Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them. Mol Cell Biol 16(7):3825–3832
Dehlin E, Wormington M, Korner CG, Wahle E (2000) Cap-dependent deadenylation of mRNA. EMBO J 19(5):1079–1086. doi:10.1093/emboj/19.5.1079
Korner CG, Wormington M, Muckenthaler M, Schneider S, Dehlin E, Wahle E (1998) The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. EMBO J 17(18):5427–5437. doi:10.1093/emboj/17.18.5427
Levy-Strumpf N, Deiss LP, Berissi H, Kimchi A (1997) DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death. Mol Cell Biol 17(3):1615–1625
Gradi A, Imataka H, Svitkin YV, Rom E, Raught B, Morino S, Sonenberg N (1998) A novel functional human eukaryotic translation initiation factor 4G. Mol Cell Biol 18(1):334–342
Yamanaka S, Zhang XY, Maeda M, Miura K, Wang S, Farese RV Jr, Iwao H, Innerarity TL (2000) Essential role of NAT1/p97/DAP5 in embryonic differentiation and the retinoic acid pathway. EMBO J 19(20):5533–5541. doi:10.1093/emboj/19.20.5533
Sugiyama H, Takahashi K, Yamamoto T, Iwasaki M, Narita M, Nakamura M, Rand TA, Nakagawa M, Watanabe A, Yamanaka S (2017) Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells. Proc Natl Acad Sci U S A 114(2):340–345. doi:10.1073/pnas.1617234114
Wu L, Fan J, Belasco JG (2006) MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103(11):4034–4039
Doench JG, Sharp PA (2004) Specificity of microRNA target selection in translational repression. Genes Dev 18(5):504–511
Pasquinelli AE, Dahlberg JE, Lund E (1995) Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases. RNA 1(9):957–967
Penman S, Rosbash M, Penman M (1970) Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. Proc Natl Acad Sci U S A 67(4):1878–1885
Hohenstein KA, Pyle AD, Chern JY, Lock LF, Donovan PJ (2008) Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells. Stem Cells 26(6):1436–1443. doi:10.1634/stemcells.2007-0857
Maess MB, Wittig B, Lorkowski S (2014) Highly efficient transfection of human THP-1 macrophages by nucleofection. J Vis Exp 91:e51960. doi:10.3791/51960
Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, Korner I, Gan L, Chen S, Castro-Obregon S, Hammermann R, Wolf J, Muller-Hartmann H, Nix M, Siebenkotten G, Kraus G, Lun K (2004) New non-viral method for gene transfer into primary cells. Methods 33(2):151–163. doi:10.1016/j.ymeth.2003.11.009
Marchenko S, Flanagan L (2007) Transfecting human neural stem cells with the Amaxa Nucleofector. J Vis Exp 6:240. doi:10.3791/240
Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17(4):438–442
Vasudevan S (2012) Functional validation of microRNA-target RNA interactions. Methods 58(2):126–134. doi:10.1016/j.ymeth.2012.08.002
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bukhari, S.I.A., Truesdell, S.S., Vasudevan, S. (2018). Analysis of MicroRNA-Mediated Translation Activation of In Vitro Transcribed Reporters in Quiescent Cells. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7371-2_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7370-5
Online ISBN: 978-1-4939-7371-2
eBook Packages: Springer Protocols