RNA activation explained

RNA activation (RNAa) is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the transcriptional/epigenetic level. RNAa was first reported in a 2006 PNAS paper by Li et al.[1] who also coined the term "RNAa" as a contrast to RNA interference (RNAi) to describe such gene activation phenomenon. dsRNAs that trigger RNAa have been termed small activating RNA (saRNA).[2] Since the initial discovery of RNAa in human cells, many other groups have made similar observations in different mammalian species including human, non-human primates, rat and mice,[3] [4] [5] [6] plant [7] and C. elegans,[8] [9] suggesting that RNAa is an evolutionarily conserved mechanism of gene regulation.

RNAa can be generally classified into two categories: exogenous and endogenous. Exogenous RNAa is triggered by artificially designed saRNAs which target non-coding sequences such as the promoter and the 3’ terminus [10] of a gene and these saRNAs can be chemically synthesized or expressed as short hairpin RNA (shRNA). Whereas for endogenous RNAa, upregulation of gene expression is guided by naturally occurring endogenous small RNAs such as miRNA in mammalian cells and C. elegans, and 22G RNA in C. elegans.  

Mechanism

The molecular mechanism of RNAa is not fully understood. Similar to RNAi, it has been shown that mammalian RNAa requires members of the Ago clade of Argonaute proteins, particularly Ago2,[11] but possesses kinetics distinct from RNAi.[12] In contrast to RNAi, promoter-targeted saRNAs induce prolonged activation of gene expression associated with epigenetic changes.[13] It is currently suggested that saRNAs are first loaded and processed by an Ago protein to form an Ago-RNA complex which is then guided by the RNA to its promoter target. The target can be a non-coding transcript overlapping the promoter[6] [11] or the chromosomal DNA.[14] The RNA-loaded Ago then recruits other proteins such as RHA, also known as nuclear DNA helicase II, and CTR9 to form an RNA-induced transcriptional activation (RITA) complex. RITA can directly interacts with RNAP II to stimulate transcription initiation and productive transcription elongation which is related to increased ubiquitination of H2B.[15] [16]

Endogenous RNAa

In 2008, Place et al. identified targets for miRNA miR-373 on the promoters of several human genes and found that introduction of miR-373 mimics into human cells induced the expression of its predicted target genes. This study provided the first example that RNAa could be mediated by naturally occurring non-coding RNA (ncRNA).[17] In 2011, Huang et al. further demonstrated in mouse cells that endogenous RNAa mediated by miRNAs functions in a physiological context and is possibly exploited by cancer cells to gain a growth advantage.[18] Since then, a number of miRNAs have been shown to upregulate gene expression by targeting gene promoters [19] [20] [21] [22] or enhancers,[23] thereby, exerting important biological roles. A good example is miR-551b-3p which is overexpressed in ovarian cancer due to amplification. By targeting the promoter of STAT3 to increase its transcription, miR-551b-3p confers to ovarian cancer cells resistance to apoptosis and a proliferative advantage.

In C. elegans hypodermal seam cells, the transcription of lin-4 miRNA is positively regulated by lin-4 itself which binds to a conserved lin-4 complementary element in its promoter, constituting a positive autoregulatory loop.[24]

In C. elegans, Argonaute CSR-1 interacts with 22G small RNAs derived from RNA-dependent RNA polymerase and antisense to germline-expressed transcripts to protect these mRNAs from Piwi-piRNA mediated silencing via promoting epigenetic activation.[25] [26]

It is currently unknown how widespread gene regulation by endogenous RNAa is in mammalian cells. Studies have shown that both miRNAs [27] and Ago proteins (Ago1) [28] bind to numerous sites in human genome, especially promoter regions, to exert a largely positive effect on gene transcription.    

Applications

RNAa has been used to study gene function in lieu of vector-based gene overexpression.[29] Studies have demonstrated RNAa in vivo and its potential therapeutic applications in treating cancer and non-cancerous diseases.[30] [31] [32] [33] [34] [35] [36]

In June 2016, UK-based MiNA Therapeutics announced the initiation of a phase I trial of the first-ever saRNA drug MTL-CEBPA in patients with liver cancer, in an attempt to activate CEBPA gene.[37] [38]

Further reading

External links

Notes and References

  1. Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R . Small dsRNAs induce transcriptional activation in human cells . Proceedings of the National Academy of Sciences of the United States of America . 103 . 46 . 17337–42 . November 2006 . 17085592 . 1859931 . 10.1073/pnas.0607015103 . 2006PNAS..10317337L . free .
  2. Li, Longcheng; Dahiya, Rajvir. "Small Activating RNA Molecules and Methods of Use." U.S. Patent US 8,877,721 filed October 1, 2004, and issued November 4, 2014.
  3. Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR . Activating gene expression in mammalian cells with promoter-targeted duplex RNAs . Nature Chemical Biology . 3 . 3 . 166–73 . March 2007 . 17259978 . 10.1038/nchembio860 .
  4. Turunen MP, Lehtola T, Heinonen SE, Assefa GS, Korpisalo P, Girnary R, Glass CK, Väisänen S, Ylä-Herttuala S . Efficient regulation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism: a novel example of epigenetherapy . Circulation Research . 105 . 6 . 604–9 . September 2009 . 19696410 . 10.1161/CIRCRESAHA.109.200774 . free .
  5. Huang V, Qin Y, Wang J, Wang X, Place RF, Lin G, Lue TF, Li LC . RNAa is conserved in mammalian cells . PLOS ONE . 5 . 1 . e8848 . January 2010 . 20107511 . 2809750 . 10.1371/journal.pone.0008848 . 2010PLoSO...5.8848H . Jin . Dong-Yan . free .
  6. Matsui M, Sakurai F, Elbashir S, Foster DJ, Manoharan M, Corey DR . Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter . Chemistry & Biology . 17 . 12 . 1344–55 . December 2010 . 21168770 . 3071588 . 10.1016/j.chembiol.2010.10.009 .
  7. Shibuya K, Fukushima S, Takatsuji H . RNA-directed DNA methylation induces transcriptional activation in plants . Proceedings of the National Academy of Sciences of the United States of America . 106 . 5 . 1660–5 . February 2009 . 19164525 . 2629447 . 10.1073/pnas.0809294106 . 2009PNAS..106.1660S . free .
  8. Seth M, Shirayama M, Gu W, Ishidate T, Conte D, Mello CC . The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression . Developmental Cell . 27 . 6 . 656–63 . December 2013 . 24360782 . 3954781 . 10.1016/j.devcel.2013.11.014 .
  9. Turner MJ, Jiao AL, Slack FJ . Autoregulation of lin-4 microRNA transcription by RNA activation (RNAa) in C. elegans . Cell Cycle . 13 . 5 . 772–81 . Jan 7, 2014 . 24398561 . 3979913 . 10.4161/cc.27679 .
  10. Yue X, Schwartz JC, Chu Y, Younger ST, Gagnon KT, Elbashir S, Janowski BA, Corey DR . Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini . Nature Chemical Biology . 6 . 8 . 621–9 . August 2010 . 20581822 . 3909968 . 10.1038/nchembio.400 .
  11. Chu Y, Yue X, Younger ST, Janowski BA, Corey DR . Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter . Nucleic Acids Research . 38 . 21 . 7736–48 . November 2010 . 20675357 . 2995069 . 10.1093/nar/gkq648 .
  12. Book: Li, Long-Cheng . https://books.google.com/books?id=r67Lrf9r9XEC&pg=PA189 . 2008 . Small RNA-mediated gene activation . Kevin V . Morris . RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity . Caister Academic Press . 978-1-904455-25-7 . 189–99.
  13. Portnoy V, Huang V, Place RF, Li LC . Small RNA and transcriptional upregulation . Wiley Interdisciplinary Reviews: RNA . 2 . 5 . 748–60 . 2011 . 21823233 . 3154074 . 10.1002/wrna.90 .
  14. Meng X, Jiang Q, Chang N, Wang X, Liu C, Xiong J, Cao H, Liang Z . Small activating RNA binds to the genomic target site in a seed-region-dependent manner . Nucleic Acids Research . 44 . 5 . 2274–82 . March 2016 . 26873922 . 4797303 . 10.1093/nar/gkw076 .
  15. Portnoy V, Lin SH, Li KH, Burlingame A, Hu ZH, Li H, Li LC . saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription . Cell Research . 26 . 3 . 320–35 . March 2016 . 26902284 . 4783471 . 10.1038/cr.2016.22 .
  16. Voutila J, Reebye V, Roberts TC, Protopapa P, Andrikakou P, Blakey DC, Habib R, Huber H, Saetrom P, Rossi JJ, Habib NA . Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer . Molecular Therapy . 25 . 12 . 2705–2714 . December 2017 . 28882451 . 5768526 . 10.1016/j.ymthe.2017.07.018 .
  17. Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R . MicroRNA-373 induces expression of genes with complementary promoter sequences . Proceedings of the National Academy of Sciences of the United States of America . 105 . 5 . 1608–13 . February 2008 . 18227514 . 2234192 . 10.1073/pnas.0707594105 . 2008PNAS..105.1608P . free .
  18. Huang V, Place RF, Portnoy V, Wang J, Qi Z, Jia Z, Yu A, Shuman M, Yu J, Li LC . Upregulation of Cyclin B1 by miRNA and its implications in cancer . Nucleic Acids Research . 40 . 4 . 1695–707 . February 2012 . 22053081 . 3287204 . 10.1093/nar/gkr934 .
  19. Matsui M, Chu Y, Zhang H, Gagnon KT, Shaikh S, Kuchimanchi S, Manoharan M, Corey DR, Janowski BA . Promoter RNA links transcriptional regulation of inflammatory pathway genes . Nucleic Acids Research . 41 . 22 . 10086–109 . December 2013 . 23999091 . 3905862 . 10.1093/nar/gkt777 .
  20. Dharap A, Pokrzywa C, Murali S, Pandi G, Vemuganti R . MicroRNA miR-324-3p induces promoter-mediated expression of RelA gene . PLOS ONE . 8 . 11 . e79467 . 2013 . 24265774 . 3827167 . 10.1371/journal.pone.0079467 . 2013PLoSO...879467D . free .
  21. Chaluvally-Raghavan P, Jeong KJ, Pradeep S, Silva AM, Yu S, Liu W, Moss T, Rodriguez-Aguayo C, Zhang D, Ram P, Liu J, Lu Y, Lopez-Berestein G, Calin GA, Sood AK, Mills GB . Direct Upregulation of STAT3 by MicroRNA-551b-3p Deregulates Growth and Metastasis of Ovarian Cancer . Cell Reports . 15 . 7 . 1493–1504 . May 2016 . 27160903 . 4914391 . 10.1016/j.celrep.2016.04.034 .
  22. Li S, Wang C, Yu X, Wu H, Hu J, Wang S, Ye Z . miR-3619-5p inhibits prostate cancer cell growth by activating CDKN1A expression . Oncology Reports . 37 . 1 . 241–248 . January 2017 . 27878260 . 10.3892/or.2016.5250 . free .
  23. Xiao M, Li J, Li W, Wang Y, Wu F, Xi Y, Zhang L, Ding C, Luo H, Li Y, Peng L, Zhao L, Peng S, Xiao Y, Dong S, Cao J, Yu W . MicroRNAs activate gene transcription epigenetically as an enhancer trigger . RNA Biology . 14 . 10 . 1326–1334 . October 2017 . 26853707 . 5711461 . 10.1080/15476286.2015.1112487 .
  24. Vaschetto LM . miRNA activation is an endogenous gene expression pathway . RNA Biology . 826–828 . April 2018 . 15 . 6 . 29537927 . 10.1080/15476286.2018.1451722 . 6152443 .
  25. Conine CC, Moresco JJ, Gu W, Shirayama M, Conte D, Yates JR, Mello CC . Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans . Cell . 155 . 7 . 1532–44 . December 2013 . 24360276 . 3924572 . 10.1016/j.cell.2013.11.032 .
  26. Wedeles CJ, Wu MZ, Claycomb JM . Protection of germline gene expression by the C. elegans Argonaute CSR-1 . Developmental Cell . 27 . 6 . 664–71 . December 2013 . 24360783 . 10.1016/j.devcel.2013.11.016 . free .
  27. Paugh SW, Coss DR, Bao J, Laudermilk LT, Grace CR, Ferreira AM, Waddell MB, Ridout G, Naeve D, Leuze M, LoCascio PF, Panetta JC, Wilkinson MR, Pui CH, Naeve CW, Uberbacher EC, Bonten EJ, Evans WE . MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression . PLOS Computational Biology . 12 . 2 . e1004744 . February 2016 . 26844769 . 4742280 . 10.1371/journal.pcbi.1004744 . 2016PLSCB..12E4744P . free .
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  30. Chen R, Wang T, Rao K, Yang J, Zhang S, Wang S, Liu J, Ye Z . Up-regulation of VEGF by small activator RNA in human corpus cavernosum smooth muscle cells . The Journal of Sexual Medicine . 8 . 10 . 2773–80 . October 2011 . 21819543 . 10.1111/j.1743-6109.2011.02412.x .
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  32. Place RF, Wang J, Noonan EJ, Meyers R, Manoharan M, Charisse K, Duncan R, Huang V, Wang X, Li LC . Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression . Molecular Therapy: Nucleic Acids . 1 . 3 . e15 . March 2012 . 23343884 . 3381590 . 10.1038/mtna.2012.5 .
  33. Yoon S, Huang KW, Reebye V, Mintz P, Tien YW, Lai HS, Sætrom P, Reccia I, Swiderski P, Armstrong B, Jozwiak A, Spalding D, Jiao L, Habib N, Rossi JJ . Targeted Delivery of C/EBPα -saRNA by Pancreatic Ductal Adenocarcinoma-specific RNA Aptamers Inhibits Tumor Growth In Vivo . Molecular Therapy . 24 . 6 . 1106–1116 . June 2016 . 26983359 . 4923325 . 10.1038/mt.2016.60 .
  34. Huan H, Wen X, Chen X, Wu L, Liu W, Habib NA, Bie P, Xia F . C/EBPα Short-Activating RNA Suppresses Metastasis of Hepatocellular Carcinoma through Inhibiting EGFR/β-Catenin Signaling Mediated EMT . PLOS ONE . 11 . 4 . e0153117 . 2016-01-01 . 27050434 . 4822802 . 10.1371/journal.pone.0153117 . 2016PLoSO..1153117H . free .
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  36. Reebye V, Huang KW, Lin V, Jarvis S, Cutilas P, Dorman S, Ciriello S, Andrikakou P, Voutila J, Saetrom P, Mintz PJ, Reccia I, Rossi JJ, Huber H, Habib R, Kostomitsopoulos N, Blakey DC, Habib NA . Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer . Oncogene . 37 . 24 . 3216–3228 . June 2018 . 29511346 . 6013054 . 10.1038/s41388-018-0126-2 .
  37. Web site: MiNA Therapeutics Announces Initiation of Phase I Clinical Study of MTL-CEBPA in Patients with Liver Cancer Business Wire. www.businesswire.com. 2 June 2016. 2016-06-06.
  38. Web site: First-in-Human Safety and Tolerability Study of MTL-CEBPA in Patients With Advanced Liver Cancer - Full Text View - ClinicalTrials.gov. clinicaltrials.gov. 2016-06-06.

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