Abstract
The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alvarez-Garcia I, Miska EA (2005) MicroRNA functions in animal development and human disease. Development 132(21):4653–4662. doi:10.1242/dev.02073
Ando T, Suzuki H, Komura K, Tanaka T, Hiraishi A, Kikuchi Y (2004) Extracellular RNAs produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum. Nucleic Acids Symp Ser (Oxf) 48:165–166. doi:10.1093/nass/48.1.165
Ando T, Suzuki H, Nishimura S, Tanaka T, Hiraishi A, Kikuchi Y (2006) Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum. J Biochem 139(4):805–811. doi:10.1093/jb/mvj091
Beckert B, Masquida B (2011) Synthesis of RNA by in vitro transcription. Methods Mol Biol 703:29–41. doi:10.1007/978-1-59745-248-9_3
Chen S, Ni M, Yu B, Lv T, Lu M, Gong F (2007) Construction and identification of a human liver specific microRNA eukaryotic expression vector. Cell Mol Immunol 4(6):473–477
Dogini DB, Pascoal VD, Avansini SH, Vieira AS, Pereira TC, Lopes-Cendes I (2014) The new world of RNAs. Genet Mol Biol 37(1 Suppl):285–293
Donohue TJ, Kaplan S (1991) Genetic techniques in Rhodospirillaceae. Methods Enzymol 204:459–485. doi:10.1016/0076-6879(91)04024-I
Dryden SC, Kaplan S (1993) Identification of cis-acting regulatory regions upstream of the rRNA operons of Rhodobacter sphaeroides. J Bacteriol 175(20):6392–6402
Du L, Pertsemlidis A (2011) Cancer and neurodegenerative disorders: pathogenic convergence through microRNA regulation. J Mol Cell Biol 3(3):176–180. doi:10.1093/jmcb/mjq058
Farrell RE Jr (2005) RNA methodologies—laboratory guide for isolation and characterization, 4th edn. Academic Press, York, USA
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811. doi:10.1038/35888
Gomes AQ, Nolasco S, Soares H (2013) Non-coding RNAs: multi-tasking molecules in the cell. Int J Mol Sci 14(8):16010–16039. doi:10.3390/ijms140816010
Hansen TA, Veldkamp H (1973) Rhodopseudomonas sulfidophila, nov. spec., a new species of the purple nonsulfur bacteria. Arch Mikrob 92(1):45–58
Hebert SS, Horre K, Nicolai L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B (2008) Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A 105(17):6415–6420. doi:10.1073/pnas.0710263105
Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ (2011) Construction and detection of expression vectors of microRNA-9a in BmN cells. J Zhejiang Univ Sci B 12(7):527–533. doi:10.1631/jzus.B1000296
Kim DY, Kim YB, Rhee YH (2000) Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida. Int J Biol Macromol 28(1):23–29. doi:10.1016/S0141-8130(00)00150-1
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Li MM, Wang WP, Wu WJ, Huang M, Yu AM (2014) Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells. Drug Metab Dispos 42(11):1791–1795. doi:10.1124/dmd.114.060145
Ling H, Fabbri M, Calin GA (2013) MicroRNAs and other non-coding RNAs as targets for anticancer drug development. Nat Rev Drug Discov 12(11):847–865. doi:10.1038/nrd4140
Liu X, Liu L, Wang Y, Wang X, Ma Y, Li Y (2014) The study on the factors affecting transformation efficiency of E. coli competent cells. Pak J Pharm Sci 27(3):679–684
Martins R, Queiroz JA, Sousa F (2010) A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography. J Mol Recognit 23(6):519–524. doi:10.1002/jmr.1078
Martins R, Queiroz JA, Sousa F (2014) Ribonucleic acid purification. J Chromatogr A 1355:1–14. doi:10.1016/j.chroma.2014.05.075
Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15(21):8783–8798. doi:10.1093/nar/15.21.8733
Nagao N, Suzuki H, Numano R, Umekage S, Kikuchi Y (2014) Short hairpin RNAs of designed sequences can be extracellularly produced by the marine bacterium Rhodovulum sulfidophilum. J Gen Appl Microbiol 60(6):222–226. doi:10.2323/jgam.60.222
Nelson PT, Wang WX, Rajeev BW (2008) MicroRNAs (miRNAs) in neurodegenerative diseases. Brain Pathol 18(1):130–138. doi:10.1111/j.1750-3639.2007.00120.x
Nilsen TW (2007) Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet 23(5):243–249. doi:10.1016/j.tig.2007.02.011
Pedro AQ, Bonifacio MJ, Queiroz JA, Maia CJ, Passarinha LA (2011) A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase. J Biotechnol 156(2):141–146. doi:10.1016/j.jbiotec.2011.08.022
Pereira P, Sousa Â, Queiroz J, Correia I, Figueiras A, Sousa F (2014) Purification of pre-miR-29b by arginine-affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 951-952:16–23. doi:10.1016/j.jchromb.2014.01.020
Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4(7):571–576. doi:10.1038/nmeth1058
Ponchon L, Dardel F (2011) Large scale expression and purification of recombinant RNA in Escherichia coli. Methods 54(2):267–273. doi:10.1016/j.ymeth.2011.02.007
Ramachandran PV, Ignacimuthu S (2013) RNA interference—a silent but an efficient therapeutic tool. Appl Biochem Biotechnol 169(6):1774–1789. doi:10.1007/s12010-013-0098-1
Sankhla IS, Bhati R, Singh AK, Mallick N (2010) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer production from a local isolate, Brevibacillus invocatus MTCC 9039. Bioresour Technol 101(6):1947–1953. doi:10.1016/j.biortech.2009.10.006
Silva F, Passarinha L, Sousa F, Queiroz JA, Domingues FC (2009) Influence of growth conditions on plasmid DNA production. J Microbiol Biotechnol 19(11):1408–1414. doi:10.4014/jmb.0805.0329
Sullenger BA, Gilboa E (2002) Emerging clinical applications of RNA. Nature 418(6894):252–258. doi:10.1038/418252a
Suzuki H, Daimon M, Awano T, Umekage S, Tanaka T, Kikuchi Y (2009a) Characterization of extracellular DNA production and flocculation of the marine photosynthetic bacterium Rhodovulum sulfidophilum. Appl Microbiol Biotechnol 84(2):349–356. doi:10.1007/s00253-009-2031-7
Suzuki H, Umekage S, Tanaka T, Kikuchi Y (2009b) Extracellular tRNAs of the marine photosynthetic bacterium Rhodovulum sulfidophilum are not aminoacylated. Biosci Biotechnol Biochem 73(2):425–427. doi:10.1271/bbb.80465
Suzuki H, Ando T, Umekage S, Tanaka T, Kikuchi Y (2010) Extracellular production of an RNA aptamer by ribonuclease-free marine bacteria harboring engineered plasmids: a proposal for industrial RNA drug production. Appl Environ Microbiol 76(3):786–793. doi:10.1128/AEM.01971-09
Suzuki H, Umekage S, Tanaka T, Kikuchi Y (2011) Artificial RNA aptamer production by the marine bacterium Rhodovulum sulfidophilum: improvement of the aptamer yield using a mutated transcriptional promoter. J Biosci Bioeng 112(5):458–461. doi:10.1016/j.jbiosc.2011.07.025
Tsutsumi A, Kawamata T, Izumi N, Seitz H, Tomari Y (2011) Recognition of the pre-miRNA structure by Drosophila Dicer-1. Nat Struct Mol Biol 18(10):1153–1158. doi:10.1038/nsmb.2125
Umekage S, Kikuchi Y (2009) In vitro and in vivo production and purification of circular RNA aptamer. J Biotechnol 139(4):265–272. doi:10.1016/j.jbiotec.2008.12.012
Wassarman KM, Zhang A, Storz G (1999) Small RNAs in Escherichia coli. Trends Microbiol 7(1):37–45. doi:10.1016/S0966-842X(98)01379-1
Wei Z, Huang W, Li J, Hou G, Fang J, Yuan Z (2007) Studies on endotoxin removal mechanism of adsorbents with amino acid ligands. J Chromatogr B Analyt Technol Biomed Life Sci 852(1–2):288–292. doi:10.1016/j.jchromb.2007.01.038
Acknowledgments
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the following projects: EXPL/BBB-BIO/1056/2012 and Pest-OE/SAU/UI0709/2014. Patrícia Pereira and Augusto Pedro acknowledge the fellowships SFRH/BD/81914/2011 and SFRH/BD/81222/2011, respectively, from FCT. The authors also acknowledge the program COMPETE (FCOMP-01-0124-FEDER-041068—EXPL/QEQ-MED/1068/2013) and the program Fundo Europeu de Desenvolvimento Regional (FEDER) [COMPETE (FCOMP-01-0124-FEDER-027560)]. The authors would like to thank Prof. Yo Kikuchi (Division of Life Science and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology) for kindly providing the pBHSR1-RM plasmid.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Electronic supplementary material
ESM 1
(PDF 922 kb)
Rights and permissions
About this article
Cite this article
Pereira, P., Pedro, A.Q., Tomás, J. et al. Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum . Appl Microbiol Biotechnol 100, 3723–3734 (2016). https://doi.org/10.1007/s00253-016-7350-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-016-7350-x