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Caenorhabditis elegans: An important tool for dissecting microRNA functions

Ziwen Zhu

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University, Boston, USA

E-mail : yjin1@bu.edu

Duo Zhang

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University, Boston, USA

Heedoo Lee

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University, Boston, USA

Yang Jin

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University, Boston, USA

DOI: 10.15761/BGG.1000106.

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Abstract

Caenorhabditis elegans (C. elegans), a member of the phylum Nematoda, carries the evolutionarily conserved genes comparing to mammals.  Due to its short lifespan and completely sequenced genome, C. elegans becomes a potentially powerful model for mechanistic studies in human diseases.  In this mini review, we will outline the current understandings on C. elegans as a model organism for microRNA (miRNA)-related research in the pathogenesis of human diseases. 

Key words

Caenorhabditis elegans, microRNA, function

Introduction

In the 1960s, Dr. Sydney Brenner first proposed using C. elegans as a model organism for the investigations focused on the neural development in animals [1].  Two decades later, the lineage of all 959 cells in the adult C. elegans were identified in Dr. John Sulston’s lab [1].  Furthermore, the first C. elegans gene was cloned, and the physical map was constructed [2].  One important milestone in biological research is that C. elegans is also the first multi-cellular organism with complete genome sequenced [3].  

C. elegans is a round worm, a member of the phylum Nematoda, and lives in the aqueous layer between the soils [1].   Its adult body only is approximate 1 mm in full length and 70 µm in diameter [4].   Bacteria (usually E. Coli strains in laboratory) serve as the food for C. elegans [5].  Interestingly, the bodies of C. elegans are transparent, providing convenience for investigators to observe the worm organs such as the intestine, gonads, etc under the microscope with no needs of staining.  Furthermore, under a high-power phase-contrast microscope, C. elegans can be observed at a single cell resolution [6].  Therefore, C. elegans is a good model to observe cell division, differentiation and death.  The life cycle of C. elegans is short.  It only takes 3-4 days to reach the adult stage [7].  The embryonic development of C. elegans is much quicker comparing to other animals.  Only after approximate 12 hours at 25°C, one can observe the evolution of each single cell through the entire development [8]. The culture of C. elegans is also convenient.  Generally, agar plates using the Petri dishes serve as a good condition for them [9].  Thus, hundreds of plates for genetic mutation screening can be easily prepared in one lab.  Conveniently, the long-term storage of C. elegans only requires -80°C freezer.  The L1 or L2 stage of worms can recover in the room temperature.  Moreover, C. elegans can be immersed in a solution containing the designated nucleic acids to acquire exogenously delivered gene modifiers, instead of requiring tedious transfection procedures.  Also, the RNA interference can be performed by feeding the C. elegans with bacteria expressing siRNA [9].

MicroRNAs (miRNAs) are a group of highly conserved noncoding RNAs and approximately 22 nucleotides in length [10].  Most of primary miRNAs (pri-miRNAs) are transcribed in the nucleus. After transcription, pri-miRNAs are then processed by microprocessor complex. Microprocessor complex binds to the stem-loop structure of pri-miRNAs and cleaves the primary transcripts to generate a hairpin-shaped RNA molecule known as precursor miRNAs (pre-miRNAs) [10]. These double-stranded pre-miRNAs are composed of 70-100 nucleotides each and subsequently transported from nucleus to the cytoplasm. Dicer is required for the mature of pre-miRNAs in the cytoplasm. The mature miRNA duplex is recognized by the RNA induced silencing complex (RISC) containing Dicer and AGO2 (argonaute RISC catalytic component 2), which are essential to miRNA-induced silencing [10].  Only one strand of miRNA duplex can be incorporated into the RISC to form miRISC, while the other strand, named miRNA* is mostly degraded.  The miRNA loaded RISC binds to the target mRNAs and silence these gene expression through either degradation of mRNA or inhibition of translation at post-transcriptional level [10].

In the process of discovering miRNAs, C. elegans played an important role as a model organism. In 1993, Lee et al. found that the gene lin-4 is not encoded for the protein, but encoded a small RNA [11]. They predicted that the lin-4 binds to the lin-14 three prime untranslated regions and downregulates the expression of LIN-14 protein. After 7 years, Reinhart et al. discovered the second miRNA involved in the development-let-7 [12]. These two miRNAs open the door of miRNA research for other species.  A variety of labs then proved that the miRNAs are present in C. elegans, Drosophila and mammalians [13-15].

miRNA functions in C. elegans

Cell death

C. elegans is an easy model to study survival and death.  C. elegans has also been used as space animals for several times. Gao et al. found 17 miRNAs which are involved in the space radiation- caused death [16]. Among these miRNAs, miR-797 is related to apoptosis by targeting ced-10 and mir-81 targets both drp-1 and hsp-1 [16]. Recent research further found that the let-7 family miRNAs, miR-48 and miR-84, have the strongest effect on ced-3, homolog of caspases 3,7 [17].

Aging

The different miRNAs can control the same target gene.  On the other hand, one miRNA can regulate a variety of target genes [18]. C. elegans can be used as a good model for this type of studies, given its relatively easy-signaling pathways. MiR-34 was the first identified miRNA up-regulating in C. elegans [19]. MiR-34 can inhibit autophagy by targeting atg-9a [20]. Lencastre et al. identified that 17 miRNAs are altered during aging processes.  Moreover, they prove that miR-71, miR-238, and miR-246 have the function of increasing longevity, demonstrated using mutant worms.  On the other hand, miR-239 can decrease life span [21].  MiR-71 interacts with the DNA damage response signaling pathways via CDC-25.1 and CHK-1 mediated pathways.  The expression of CDC-25.1 in aged miR-71 knock-out worms is increased by 8-folds [21]. Despite that several screenings have suggested that the aging process relates to miRNAs, the mechanisms underlying the miRNA-mediated regulation of longevity remain to be further determined [21,22].

Metabolism

Dietary restriction (DR) is the most effective way to reduce age-related phenotypes and to extend lifespan, it can also promote longevity and protect against age-associated disease across species. C. elegans is a great model to examine the molecular mechanisms by which miRNAs coordinate food intake with health-promoting metabolism [23]. Vora et al. found that mir-80 is a major regulator for the DR state [24]. MiR-80 knock-out worms maintain cardiac and skeletal muscle-like function at older age, reduce accumulation of lipofuscin, and extend lifespan.  These functions of miR-80 is very similar to the physiological features of DR [24]. With food limitation, decreased miR-80 levels upregulate CBP-1 protein levels to engage metabolic loops that promote DR [24]. Also, miR-71 and miR-228 mediate dietary-restriction-induced longevity [25]. Furthermore, PHA-4 and SKN-1 are negatively regulated by miR-228.  On the other side, miR-71 represses PHA-4 [25].

Innate immunity

The let-7 family is initially found relating to the innate immunity of C. elegans [26]. The let-7 family mutant worms are more resistant to pathogen infections by downregulating of heterochronic genes and the p38 MAPK pathways [26]. The developmental timing signal or ATF-7 regulate hbl-1 to control seam cell proliferation via regulating miR-48, miR-84 and miR-241 [26].

Development

C. elegans is an easy model to study the development, given that it only takes 2-3 days for this round worm to grow from eggs to adults.  Karp et al. identified 14 miRNAs which are related the development of C. elegans.  Most development miRNAs are altered during the stages from L2 to  L2d or L2 molt to dauer [27]. The second found miRNA, let-7, has an important role in embryonic developing [28]. Let-7 regulates the transition process from L4 stage to adult stage.  Without let-7, worms can’t grow into mature stage due to uncontrolled downregulation of hbl-1, lin-4 and daf-12 [12,29].  Further studies found that let-7 family members miR-48, miR-84, and miR-241 all regulate developmental timing from the stage L2 transit into L3.  The hbl-1 is also a target similar to let-7 [29].  MiR-51 family contains miR-51, miR-52, miR-53, miR-54, miR-55, and miR-56.  This family has also been proved involved in the early development stage.  Mutant worms of these miRNAs have the phenotypes of unattached penetrant pharynx [30,31].  Additionally, CDH-3, the mammalian fat cadherin homolog,  is the target of the miR-51 family miRNAs [30].

Stress response

The miR-360 knock-out worms were also proved to increase the function of glycyrrhizic acid [32]. And the same group prove that graphene oxide can regulate miR-360 to decrease DNA damage-apoptosis signaling by targeting CEP-1 [33,34]. Also miR-355 could regulate MWCNTs toxicity by target daf-2, the gene encodes for the insulin-like growth factor 1 [35] (Table 1).

Table 1. miRNAs in C. elegans with known functions

miRNA family

MiRNA name

Function

References

let-7

let-7

DNA damage response, mitochondrial aging DNA damage response, mitochondrial respiration, germline maintenance

[21,22,36]

let-7

miR-48

Death, development

[17,21]

let-7

miR-84

Death, development

[17,21]

let-7

miR-241

Development

[19,21]

lin-4

lin-4

Aging, stress response

[37]

miR-2

miR-797

Death

[16]

miR-34

miR-34

Aging, DNA damage response, Cell death, radiosensitive

[38]

miR-51

miR-51

Development

[30,31]

miR-51

miR-52

Development

[30,31]

miR-51

miR-53

Development

[30,31]

miR-51

miR-54

Development

[30,31]

miR-51

miR-55

Development

[30,31]

miR-51

miR-56

Development

[30,31]

miR-71

miR-71

Aging, DNA damage response, germline signaling, metabolism

[39]

miR-80

miR-81

Aging, death, metabolism

[16]

miR-228

miR-228

Aging, metabolism

[21,25]

miR-238

miR-238

Aging

[21]

Conclusions and perspectives

C. elegans has been used as a model organism for more than 50 years, its biological properties facilitate the miRNA research robustly.  That being said, there remain many uncertainties and undiscovered functions in the field of C. elegans miRNA research which require further investigations. Current research suggests that a lot of miRNAs was involved in development, cell death, aging, innate immunity, metabolism, and stress response. But further research is needed to identify the functions and detailed signaling pathways to better understand the systems-level role of miRNAs.

Acknowledgement

This work is support by NIH R01HL102076 (Y.J.), NIH R21 AI121644 (Y.J.) and NIH R01 GM111313 (Y.J.).

References

  1. Kitano H, Hamahashi S, Luke S (1998) The perfect C. elegans project: an initial report. Artif Life 4: 141-156. [Crossref]
  2. Coulson A, Sulston J, Brenner S, Karn J (1986) Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 83: 7821-7825. [Crossref]
  3. C. elegans Sequencing Consortium. (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282: 2012-2018. [Crossref]
  4. Petzold BC, Park SJ, Ponce P, Roozeboom C, Powell C, et al. (2011) Caenorhabditis elegans body mechanics are regulated by body wall muscle tone. Biophys J 100: 1977-1985. [Crossref]
  5. Abada EA, Sung H, Dwivedi M, Park BJ, Lee SK, et al. (2009) C. elegans behavior of preference choice on bacterial food. Mol Cells 28: 209-213. [Crossref]
  6. Chen BC, Legant WR, Wang K, Shao L, Milkie DE, et al. (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346: 1257998. [Crossref]
  7. Johnsen RC, Calrk DV (1989) Mapping Genes in C. elegans. Tested studies for laboratory teaching 109.
  8. Porta-de-la-Riva M, Fontrodona L, Villanueva A, Cerón J (2012) Basic Caenorhabditis elegans methods: synchronization and observation. J Viz Exp: e4019. [Crossref]
  9. Frézal L, Félix MA (2015) C. elegans outside the Petri dish. Elife 4. [Crossref]
  10. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15: 509-524. [Crossref]
  11. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-854. [Crossref]
  12. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, et al. (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403: 901-906. [Crossref]
  13. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294: 853-858. [Crossref]
  14. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294: 858-862. [Crossref]
  15. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294: 862-864. [Crossref]
  16. Gao Y, Li S, Xu D, Wang J, Sun Y (2015) Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission. J Radiat Res 56: 872-882. [Crossref]
  17. Weaver BP, Zabinsky R, Weaver YM, Lee ES, Xue D, et al. (2014) CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in C. elegans. Elife 3: e04265. [Crossref]
  18. Krek A, Grün D, Poy MN, Wolf R, Rosenberg L, et al. (2005) Combinatorial microRNA target predictions. Nat Genet 37: 495-500. [Crossref]
  19. Inukai S, Slack F (2013) MicroRNAs and the genetic network in aging. J Mol Biol 425: 3601-3608. [Crossref]
  20. Yang J, Chen D, He Y, Meléndez A, Feng Z, et al. (2013) MiR-34 modulates Caenorhabditis elegans lifespan via repressing the autophagy gene atg9. Age (Dordr) 35: 11-22. [Crossref]
  21. de Lencastre A, Pincus Z, Zhou K, Kato M, Lee SS, et al. (2010) MicroRNAs both promote and antagonize longevity in C. elegans. Curr Biol 20: 2159-2168. [Crossref]
  22. Kato M, Chen X, Inukai S, Zhao H, Slack FJ (2011) Age-associated changes in expression of small, noncoding RNAs, including microRNAs, in C. elegans. RNA 17: 1804-1820. [Crossref]
  23. Fontana L, Partridge L, Longo VD (2010) Extending healthy life span--from yeast to humans. Science 328: 321-326. [Crossref]
  24. Vora M, Shah M, Ostafi S, Onken B, Xue J, et al. (2013) Deletion of microRNA-80 activates dietary restriction to extend C. elegans healthspan and lifespan. PLoS Genet 9: e1003737. [Crossref]
  25. Smith-Vikos T, de Lencastre A, Inukai S, Shlomchik M, Holtrup B, et al. (2014) MicroRNAs mediate dietary-restriction-induced longevity through PHA-4/FOXA and SKN-1/Nrf transcription factors. Curr Biol 24: 2238-2246. [Crossref]
  26. Ren Z, Ambros VR (2015) Caenorhabditis elegans microRNAs of the let-7 family act in innate immune response circuits and confer robust developmental timing against pathogen stress. Proc Natl Acad Sci U S A 112: E2366-E2375. [Crossref]
  27. Karp X, Hammell M, Ow MC, Ambros V (2011) Effect of life history on microRNA expression during C. elegans development. RNA 17: 639-651. [Crossref]
  28. Roush S, Slack FJ (2008) The let-7 family of microRNAs. Trends Cell Biol 18: 505-516. [Crossref]
  29. AL Abbott, E Alvarez-Saavedra, EA Miska, NC Lau, DP Bartel, et al. (2005) The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev cell: 403-414. [Crossref]
  30. WR Shaw, J Armisen, NJ Lehrbach, EA Miska (2010) The conserved miR-51 microRNA family is redundantly required for embryonic development and pharynx attachment in Caenorhabditis elegans. Genetics, 185: 897-905. [Crossref]
  31. Alvarez-Saavedra E, Horvitz HR (2010) Many families of C. elegans microRNAs are not essential for development or viability. Curr Biol 20: 367-373. [Crossref]
  32. Zhao Y, Jia R, Qiao Y, Wang D (2016) Glycyrrhizic acid, active component from Glycyrrhizae radix, prevents toxicity of graphene oxide by influencing functions of microRNAs in nematode Caenorhabditis elegans. Nanomedicine 12: 735-744. [Crossref]
  33. Zhao Y, Wu Q, Wang D (2016) An epigenetic signal encoded protection mechanism is activated by graphene oxide to inhibit its induced reproductive toxicity in Caenorhabditis elegans. Biomaterials 79: 15-24. [Crossref]
  34. Zhao Y, Wu Q, Wang D (2015) A microRNAs–mRNAs network involved in the control of graphene oxide toxicity in Caenorhabditis elegans. RSC Advances 5: 92394-92405.
  35. Zhao Y, Yang J, Wang D (2016) A MicroRNA-Mediated Insulin Signaling Pathway Regulates the Toxicity of Multi-Walled Carbon Nanotubes in Nematode Caenorhabditis elegans. Sci Rep 6: 23234. [Crossref]
  36. C Ibáñez-Ventoso, M Yang, S Guo, H Robins, RW Padgett, M Driscoll (2006) Modulated microRNA expression during adult lifespan in Caenorhabditis elegans. Aging cell 5: 235-246. [Crossref]
  37. Boehm M, Slack F (2005) A developmental timing microRNA and its target regulate life span in C. elegans. Science 310: 1954-1957. [Crossref]
  38. Kato M, Paranjape T, Müller RU, Nallur S, Gillespie E, et al. (2009) The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells. Oncogene 28: 2419-2424. [Crossref]
  39. Boulias K, Horvitz HR (2012) The C. elegans microRNA mir-71 acts in neurons to promote germline-mediated longevity through regulation of DAF-16/FOXO. Cell Metab 15: 439-450. [Crossref]

Editorial Information

Editor-in-Chief

Hieronim Jakubowski

Rutgers University-New Jersey Medical School

Article Type

Mini Review

Publication history

Received: June 02, 2016
Accepted: June 22, 2016
Published: June 25, 2016

Copyright

©2016 Zhu Z. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Zhu Z, Zhang D, Lee H, Jin Y (2016) Caenorhabditis elegans: An important tool for dissecting microRNA functions. Biomed Genet Genomics 1: DOI: 10.15761/BGG.1000106.

Corresponding author

Yang Jin, MD, Ph.D.

Division of Pulmonary and Critical Care, Department of Medicine, Boston University Medical Campus, 72 East Concord St., Boston, MA 02118, USA.

E-mail : yjin1@bu.edu

Table 1. miRNAs in C. elegans with known functions

miRNA family

MiRNA name

Function

References

let-7

let-7

DNA damage response, mitochondrial aging DNA damage response, mitochondrial respiration, germline maintenance

[21,22,36]

let-7

miR-48

Death, development

[17,21]

let-7

miR-84

Death, development

[17,21]

let-7

miR-241

Development

[19,21]

lin-4

lin-4

Aging, stress response

[37]

miR-2

miR-797

Death

[16]

miR-34

miR-34

Aging, DNA damage response, Cell death, radiosensitive

[38]

miR-51

miR-51

Development

[30,31]

miR-51

miR-52

Development

[30,31]

miR-51

miR-53

Development

[30,31]

miR-51

miR-54

Development

[30,31]

miR-51

miR-55

Development

[30,31]

miR-51

miR-56

Development

[30,31]

miR-71

miR-71

Aging, DNA damage response, germline signaling, metabolism

[39]

miR-80

miR-81

Aging, death, metabolism

[16]

miR-228

miR-228

Aging, metabolism

[21,25]

miR-238

miR-238

Aging

[21]