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RNA interference in free living flatworms

RNA interference in free living flatworms

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An essay for the 2011 Undergraduate Awards (Lecturer Nomination) Competition by Thomas Fleming. It is nominated by Lecturer David Timson of Queen's University Belfast in the category of Life Sciences
An essay for the 2011 Undergraduate Awards (Lecturer Nomination) Competition by Thomas Fleming. It is nominated by Lecturer David Timson of Queen's University Belfast in the category of Life Sciences

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Published by: Undergraduate Awards on Sep 01, 2012
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10/27/2013

 
RNA interference in free living flatworms
Introduction
RNA interference (RNAi) has become an increasingly popular research tool that inducespost transcriptional gene silencing through the introduction of double stranded RNA(dsRNA) and the initiation of a cascade of cellular events.The research of Fire and Mello (Fire
et al 
., 1998) was the first to suggest anddemonstrate that dsRNA was the initial trigger for RNAi in
Caenorhabditis elegans
. Thestudy was influenced by previous work on antisense mediated silencing by Guo andKemphues (1995) where both antisense RNA , and the then believed control senseRNA, induced silencing. Fire’s study hypothesised that dsRNA formed in minute molar concentrations in the separate sense or antisense solutions and proved that lowconcentrations of dsRNA formed within the mixture of sense and antisense solutionsand was the key trigger in gene silencing. Thereafter many more organisms have beenshown to be amenable to RNAi, these include
Drosophila melanogaster 
(Kennerdell andCarthew, 1998) and
Trypanosoma brucei 
(Ngo
et al 
., 1998).Since the acknowledgment of dsRNA as ‘potent’ initiator for RNAi, many biological andbiomedical researchers have used dsRNA to silence the expression of specific genesassociated with infectious diseases and neurodegenerative disorders (Mocellin andProvenzano, 2004). Equally significant has been the role of RNAi in investigating genefunction.Free living flatworms (Platyhelminthes from the class Turbellaria) have been extensivelystudied for over a century, particularly due to their strong regenerative abilities(Palakodeti
et al 
., 2006). Although these organisms have been described over such anextended period of time, genetic manipulation has only been attempted in relativelyrecent studies. The ability to easily maintain free living flatworms
in vitro
has beensignificant in developing their role as model organisms, as they share many similar traitswith the three parasitic platyhelminth classes that are more difficult to maintain inlaboratory conditions. Here we discuss the mechanisms of RNAi and its use in freeliving flatworms.
Mechanisms of RNAi
The RNAi process relies on two classes of small RNA molecules which were discoveredafter the initial silencing research by Fire
et al 
. These molecules were short interferingRNA (siRNA) (Hamilton and Baulcombe, 1999) and micro RNA (miRNA) (Ambros
et al 
.,2003).
 
The siRNA pathways outlined below have been reviewed by Naqvi
et al 
. (2009). siRNAsare ~20-24 nucleotides long and result from mechanisms induced specifically to preventthe activity of exogenous viral nucleic acids and maintain genome integrity by silencingunwanted loci such as retrotransposonsThe siRNA pathway in animals begins with a precursor dsRNA molecule produced bytranscription or by an enzymatic reaction involving single stranded RNA. RNaseIIIendonuclease (Dicer) then cleaves the dsRNA forming the siRNA molecules.Subsequently the siRNA Dicer complex transports into the cytoplasm where thehelicase Argonaute (an RNA binding protein) is recruited (Zhou
et al.,
2007). This in turngives rise to the RNA induced silencing complex (RISC) that facilitates binding anddestruction of the target complementary mRNA (Sen and Blau, 2005) thus inhibiting theproduction of the encoded protein. Another stage of siRNA biogenesis is the triggeringof an amplification cascade that involves the production of secondary siRNAs derivedfrom RNA directed RNA polymerase (RdRP). The belief is that RdRP produces siRNAin response to the presence of existing siRNAs in the cell (Sijen
et al.,
2001).
Figure 1. siRNA pathway (after Naqvi
et al 
., 2009)
MicroRNAs are a class of regulatory molecules also involved in RNAi processes. Approximately 18-24 nt long, miRNAs are non-protein coding RNA molecules encodedin the genome and were first observed in a
C. elegans
study on gene lin-4 (Lee
et al 
.,
 
1993) and then later classified as miRNA. This class of dsRNAs are crucial inendogenous gene expression, either through degradation of the mRNA or by inhibitionof translation. Studies have observed miRNAs involved in determining cell function andlineage. The work of Yekta
et al 
. (2004) indicated miRNA-mediated post transcriptionoccurred on HOX genes which control segment arrangement in early embryonic stagesof metazoans.The biogenesis of miRNA involves more than one pathway (Baulcombe, 2004). Thestandard miRNA pathway in animals begins with transcription of a hairpin structure pri-miR by RNA polymerase polII. Post-transcriptional modification begins with theconversion of pri-miR to the distinctive stem loop structure pre-miR by the RNaseIIIenzyme Drosha and cofactor Pasha. Pre-miR is transported into the cytoplasm assistedby Exportin-5 (Yi
et al 
. 2003) where Dicer cleaves the pre-miR, generating miRNAduplexes which bind with micro Ribo Nucleo Protein (mi-RNP). The mi-RNP/ miRISCthen facilitate binding to the target mRNA and leads to translation silencing activation or silencing. The silenced mRNAs are stored in organelle P-bodies which contain proteinsused in miRNA degradation (Kulkarni
et al 
., 2010).
Figure 2. miRNA pathway
 
(after Naqvi
et al 
., 2009)

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