International Journal Of Biology Volume 1, Issue 2  Effective and certain inhibition of microRNAs             in Caenorhab...
International Journal Of Biology Volume 1, Issue 2Abstract:MicroRNAs (miRNAs) are little non-coding RNAs that will control...
International Journal Of Biology Volume 1, Issue 2Although such mutant strains happen to be        Nathan Barry and co-wor...
International Journal Of Biology Volume 1, Issue 2shown by let-7 knockout mutant animals [5].      Overall, the data prese...
International Journal Of Biology Volume 1, Issue 2References:                                            R: MicroRNA-143 r...
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Effective and certain inhibition of microRNAs in Caenorhabditis elegans


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Caenorhabditis elegans is a free-living, transparent nematode (roundworm), about 1 mm in length, which lives in temperate soil environments.

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Effective and certain inhibition of microRNAs in Caenorhabditis elegans

  1. 1. International Journal Of Biology Volume 1, Issue 2 Effective and certain inhibition of microRNAs in Caenorhabditis elegans By: Nathan Page 1
  2. 2. International Journal Of Biology Volume 1, Issue 2Abstract:MicroRNAs (miRNAs) are little non-coding RNAs that will control the manifestation of several aim forgenes. Yet, while 100s of miRNAs have already been acknowledged, little is known with regards to theirfunctions. In a very recent report unveiled by, Nathan Barry and his co-workers they demonstrated aprocess for powerful as well as particular knockdown of miRNA manifestation in Caenorhabditis elegansby using altered antisense oligonucleotides, that may be applied like a effective tool for study regardingregulation and performance of miRNAs in vivo.MicroRNAs (miRNAs) are roughly 22- Although animals like Caenorhabditisnucleotidelong non-coding RNA molecules elegans contain countless miRNAs, little isthat had been first discovered in known with regards to functions.Caenorhabditis elegans but they are now Additionally, using the development of deepregarded as being expressed in most sequencing technology, novel miRNAs aremetazoans. miRNA genes can be located increasingly being discovered in a variety ofsingly or even in groupings, frequently using model microbes, requiring faster plus muchtheir individual and regulatory elements, more convenient techniques to examine theireven though some miRNA genes may also functional importance through controllingbe present within the introns of other protein their activity in vivo.coding genes.miRNAs are transcribed as extended major A present report by Nathan and associates,transcripts and tend to be prepared first [2] now illustrates, the effective and specificwithin the nucleus through the In hibition of miRNAs, in Caenorhabditismicroprocessor DRSH-1/DROSHA as well elegans by dextran-conjugated antisenseas in the cytoplasm via the DCR-1/DICER oligonucleotides. miRNAs had been firstcomplex to produce the active 21 to 23- identified inCaenorhabditis elegans in 1993nucleotide RNA molecules. [3]They are utilized through the RNA-caused Caenorhabditis elegans is in fact useful forsilencing complex (RISC) and led towards those who analyze it because of it is easythe 3’ untranslated areas of target messenger genetics, totally sequenced genome as wellRNAs (mRNAs) with complete or as simple anatomy. To look at the objectiveunfinished collection complementarity, of person miRNAs in a assortment ofcausing in either degradation from the target cellular pathways, forward geneticsmRNA or inhibition of their translation. approaches have created many strains.[4,5]Just one miRNA can target multiple mRNAs Although such mutant strains happen to bewhich capacity to control the expression of extensively employed for functional studiesseveral genes makes miRNAs pivotal in from the target miRNAs, their generation iscontrolling numerous existence processes, both time intensive and labor intensive.from development as a result of metabolic Further, Its tiresome to create knockoutprocesses to senescence, maturing and alleles for miRNAs which are required fordeclining (examined in [1]). survival and Page 2
  3. 3. International Journal Of Biology Volume 1, Issue 2Although such mutant strains happen to be Nathan Barry and co-workers [2] conjugatedextensively employed for functional studies 2’-oxy-methyl antisense oligonucleotidesfrom the target miRNAs, their generation is complementary towards the target miRNAsboth time intensive and labor intensive. using the polysaccharide dextran, that hasFurther, its tiresome to create knockout high solubility in water and shows elevatedalleles for miRNAs which are required for cellular uptake and availability. The authorssurvival and development, and in instances also modified these oligonucleotides bywhere the miRNAs are situated within the conjugating 1 to 3 molecules of rhodamineintronic sequences of protein coding genes, per molecule of dextran. These modifiedits possible their knockout will perturb antisense oligonucleotides were injected inexpression from the protein coding gene. to the syncytial gonads of adult earthworms and embryos were selected in line with theIts also hard to particularly get rid of just existence of rhodamine (Figure 1).one miRNA from the miRNA gene clusterwithout having affected the expression fromthe remaining miRNAs within the cluster.Many groups have attempted using reversegenetics methods to hinder specific miRNAfunction transiently in various modelsystems. Typically the most popular tool ofpreference is in a different way modifiedantisense oligonucleotides, which are simpleto synthesize and deliver.Several research groups have showninhibition of miRNA function with limitedsuccess using antisense oligonucleotides forexample locked nucleic chemicals (modifiedRNA nucleotides) [6] or morpholinos(nucleic acidity analogs) [7] in Drosophila,zebrafish, and rodents [8-10].C. elegans continues to be broadly usedbecause the model system to review theorganic role of small non-coding RNAs but,thus far, no standard procedures ortechniques are for sale to effectively andeasily knockdown miRNA functiontransiently. To hinder miRNA expression in Nathan Barry and associates [2] utilizedC. elegans, Zamore’s group injected 2’- antisense oligonucleotides supporting to lin-oxymethyl oligonucleotides into developing 4 and let-7 For example, progeny of animalsembryos [11]. injected with antisense oligonucleotides.Their embryo injection strategy is The differentiation of seam cells and lack oftechnically difficult and, therefore, is not vulva formation, known phenotypes for lin-used extensively. In addition, the main 4(lf ) [3]. The progeny of worms injecteddisadvantage to with such modified with antisense oligonucleotidesoligonucleotides may be the incumbent complementary to let-7 demonstrated atoxicity triggered by poor solubility and bursting vulva phenotype with lack of alaeinsufficient cytoplasmic retention and tissue (longitudinal ridge) formation in 100% ofdistribution. To deal with these problems, animals, consistent with the Page 3
  4. 4. International Journal Of Biology Volume 1, Issue 2shown by let-7 knockout mutant animals [5]. Overall, the data presented in the report byBy contrast, progeny of animals treated with Nathan and [2] convincingly suggest thatantisense oligonucleotides complementary this new class of antisense oligonucleotidesto miR-84, a let-7 family member, exhibited could prove to be a very powerful andno visible phenotype, consistent with the effective tool for the robust and specificknown phenotype of mir-84(lf ) mutants. inhibition of miRNA function in vivo. miR-84 includes a sequence that is highly This technique would be very useful forhomologous to let-7 sequence, yet the studying the different biological functions ofphenotype observed in the miR-84 essential and novel miRNAs in C. elegans.knockdown animals was completely unlike While it remains to be tested, thisthe let-7 knockdown phenotype therefore technology does not appear to be limited toconfirming the specificity of inhibition. just C. elegans, and could be applicable in other organisms as well.Using a similar strategy, the experts maypossibly exhibit specific inhibition of lys-6miRNA in the ASEL neurons, where itfunctions to maintain left-right symmetry.This technique also successfullydemonstrated the inhibition in activity ofmiR-42, which is active only duringembryonic development.In addition, the authors demonstrate that thisnew class of antisense oligonucleotidescould be utilized for specific andsimultaneous inhibition of two miRNAs, inthis case lin-4 and lys-6.As with all antisense techniques, thedextranconjugated antisense 2’-oxy-methyloligonucleotides have a limitation, in thiscase with respect to the period of efficacy ofinhibition against target miRNAs in theanimal.The inhibition was robust until 15-20 hoursafter the L4 molt with higher doses ofinjection, after which the effect diminished,most probably due to the dilution of theantisense oligonucleotides inside the animal.For this reason this method of miRNAinhibition might not be the most effectivefor long-term functional assays, such asaging assays, where the miRNA in questionneeds to be inhibited throughout the lifespan of the Page 4
  5. 5. International Journal Of Biology Volume 1, Issue 2References: R: MicroRNA-143 regulates adipocyte differentiation. J Biol Chem 2004,  Esquela-Kerscher A, Slack FJ: 279:52361-52365. Oncomirs – microRNAs with a role in cancer. Nat Rev Cancer 2006, 6:259-  Hutvágner G, Simard MJ, Mello CC, 269. Zamore PD: Sequence-specific inhibition of small RNA function. PLoS  Zheng G, Ambros V, Li W: g miRNA in Biol 2004, 2:e98. doi:10.1186/jbiol230 Caenorhabditis elegans using a potent Cite this article as: Kundu ST and Slack and selective antisense reagent. Silence FJ: Robust and specific inhibition of 2010, 1:9 microRNAs in Caenorhabditis elegans. Journal of Biology 2010, 9:20.  Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993, 75:843-854.  Miska EA, Alvarez-Saavedra E, Abbott AL, Lau NC, Hellman AB, McGonagle SM, Bartel DP, Ambros VR, Horvitz HR: Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. PLoS Genet 2007, 3:e215.  Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G: The 21- nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000, 403:901-906.  Orom UA, Kauppinen S, Lund AH: LNA- modified oligonucleotides mediate specific inhibition of microRNA function. Gene 2006, 372:137-141.  Flynt AS, Li N, Thatcher EJ, Solnica- Krezel L, Patton JG: Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nat Genet 2007, 39:259-263.  Boutla A, Delidakis C, Tabler M: Developmental defects by antisensemediated inactivation of micro- RNAs 2 and 13 in Drosophila and the identification of putative target genes. Nucleic Acids Res 2003, 31:4973-4980.  Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M: Silencing of microRNAs in vivo with ‘antagomirs’. Nature 2005, 438:685-689.  Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett CF, Lollo B, Page 5