MiRNA is a recently discovered new class of small RNA molecules which were first discovered in caenorhabtidis elegans (Lee et al. 1993). MiRNA splays a significant role in the prediction of the gene and protein expression.The discovery of MiRNA has also been called as 'Tip of the iceberg' and 'small RNA revolution'.This brings us to the fact that though we have had an wide knowledge of the gene revolution ,we never really knew the importance of MiRNA.(Lee et al. 1993)This helps us in knowing a new branch in gene regulation .They are also involved in a variety of processes like cell proliferation and apoptosis ,development,differentiation senesence and metabolism and immunity in a variety of organism. miRNA are conserved in most of the organism and more than 650 miRNA are clsasified from the Human genome.When it comes to Drosophila melanogaster about 40 miRNA are descovered from the whole Genome.The most important part of which is that approximately 30% of the genes were found to be protein coding genes which means most of the miRNa are believed to be found in the protein coding genes.
Fig1 Secondary structure of pre miRNA. Predicted targeting of the D. melanogaster gene fushi-tarazu by mir-3. Shown is the mRNA/miRNA duplex as predicted by MFOLD. Draft Introduction Kitson Davidson mony
The free energy is −30.8 kcal/mol, the nucleus has a score of 28 (P value 0.0001) and is located at the 5′ end of the miRNA and 173 bases downstream of the stop codon. Another putative target site for mir-3 is located 99 bases further downstream. The fushi-tarazu ortholog in pseudoobscura also has a predicted target site for mir-3. All nuclei are found close to the 5′ end of the miRNA. GC base pairings are marked in red, AU and GU in blue.(Grun et al. 2005)
Through out the years we have learnt that miRNA are a class of Rna derived from a prototype of RNA ,the siRNA.Nearly every metazoans miRNa is classified
miRNAs are short single stranded RNA of of 22-24 nucleotide long transcribed from non coding regions.This process is carried out by RNA polymerase Iithrough the long pre-miRNA structures of 60-70 nucleotide hairpin structure.which is seperated by a single stranded RNA.(Ambros 2004)
It is also known that the regulation of gene differsbased on the various complexes that are formed with their respective targets.Based on their binding that is either perfect base pairing or imperfect basepairing their pathways differ.When miRNA binds with the mRNA then the perfect binding occurs were mRNA cleavage occurs through silencing mechanism.When miRNA binds to MiRISC then imperfect binding occurs which leads to repression of translation.(Pegoraro & Tauber 2008)
Although miRNA are known for a recent number of years for their role in Downregulation of gene they have also been known recently for their increase in post translation.(Vasudevan et al. 2007)
When the microarray analysis of Drosophila heads were observed then 78 miRNAs were expressing the light-dark circle and they were compared with the clock mutant cycle.miR263a and mir263b both showed significant cycling. Their expression when profiled using a qPCR then it is known that these genes cycled continuously in Dark cycles.SInce their fold recognition is very small this can be ignored however when the whole heads of drosophilla was set to microarray analysis it was found that these genes helped in changing the individual neuron at a much higher level .
Using the different prediction algorithm various targgets were found which might be used as a target for the circadian miRNAs.
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Apart from miRNAs being involved in daily cycles they are also involved in seasonal changes for example in A.thaliana the circadian genes which are involved in seasonal flowering .
Apart from this miRNa alterations are also found to be involved in the initiation and progression of human cancer miRNa expression profiling of human tumors has identified signatures involved in diagnosis , staging, progression,prognosis and response to treatment.In addition MiRNA profiling also suggests that the mi RNA genes which represents Downstream target of oncogenic pathways or the protein coding genes which is involved in cancer.(Calin & Croce 2006)
These genes which are expressed abnormally in human cancers target transcripts of essential protein coding genes for example.Ras oncogenes by let-7 family memebers,the BCL2 anti apoptic gene by the mir-15a-mir-16-1 cluster,the E2F1 transcription factor by the mir-17-92 cluster or the BCL6 apoptotic gene by mir127.(Calin & Croce 2006)
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The ctf(circadian transcription factors) induce the transcription of miRNA genes .the protein complex processes the pri-miRNA genes and produces a 60-70nt pre miRNA.Exportin transclocates it and Dicer cuts the pre miRNA and delivers the miRNA duplex .The miRNa when loaded with RISc imperfect base pairing takes place which leads to inducing mi-RISC-dependent inhibition and translation. While when miRNA is loaded with mRNA perfect cleavage is seen(si-RISC dependent silencing). (Pegoraro & Tauber 2008)
Our research involves Drosophila melanogaster,Traditionally Drosophila has been used in biological research particularly in genetics and developmental biology because they can be easily bred and maintained at a low cost .It is one of the most widley used organism and one of the best Eukaryote to study . Most of the eukaryotes use the general process such as transcription and replication and so do humans , this helps us in human studies by comparison.About 75% of the known human disease genes are expressed in the flies genetic code. (Reiter et al. 2001)
An online Database called as Homophila is available to search for human disease gene homologues were for most of the human genetic diseases Drosophila is used as a model It is been used in Parkinsons ,Huntingtons.spinocerebellar ataxia and alzheimers disease.Apart from the genes eing similar 50% of the fly protein sewuences have mamalian analogues.(Reiter et al. 2001)
MicroRNAs act as endogenous translational repressors of protein coding genes in animals by binding to target sites in the 3'UTRs of mRNAs.A single nucleotide change in the sequence can affect the miRNA regulation ,naturally occuring SNPs in target sites are candidates for functional variation.This is mainly useful for studying their biomedical application and evolutionary studies related to the organism.The same applies to Flies and since they are all eukaryotes comparitive studies can be taken place.The amount of studies taken for flies in regard with the variation is very low and regarding the miRNA target sites.
Approximately 250 SNPs potentially create novel target sites for miRNAs in humans.If
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some variants have functional effect they might confer phenotypic differencec among humans.Although they appear to be evolving under neutrality some of these SNPs are found at high population frequencies even in experimentally verified targets or long range haplotypes that may have been subject to recent positive selection.(Reiter et al. 2001, Saunders et al. 2007)
Alleles for a human SNP may create a target site that is not evolutionarily conserved .A 3'UTR can create a sequence match to to the seed of the miRNA that was not priously matched with the given mRNA.However this is not Biologically relevant until the appropriate cognate miRNA is coexpressed both temporally and spatially witht the given mRNa.
Although widespread extent by microRNA is comparable between insects and animals but that specific micro RNA can be clade specific that is they are fuctional based on their species . For example, mir 210 which is an Fly specific micro Rna which helps during the regulation of oogenesis. Through High throughput sequencing methods it was found that approximately eight targets per Micro Rna was found however it is estimated way higher than this.
For our research we used the target list given by the UCSC genome browser .This had the miRNA target database onli for April 2004.This was derived from pictar algorithm. The prediction is based on the picTar score the higher the score the better the prediction .Apart from using the Pictar score they have also used the result from the comparison of seven Drosophilla species for accuracy.
Sanger also provides this Target list which is not so perfectly screened . The amount of targets given by Sanger is almost 38,772 and also They used the Pvalue for the prediciton .The accuracy is greatly reduced by this process. While the Ucsc browser provided a small data set of approximately 24103 .
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The target list acquired from UCSC browser where the second row shows the gene location and the start and end rows ahows the coordinates of the miRNA.
(http://hgdownload.cse.ucsc.edu/goldenPath/dm2/database/)
The first step would be to remove all those rows which have coordinates of length more than 50nt in length .For this we have to first add 20 to the end row and substract 20 for the positive section this is done because, expression assays have shown that miRNa genes that are located some 50 nt of each other are often coexpressed.
So after choosing those target list which came around 7177 were taken and their coordinates and their genomic location was used to extract the sequences. For extracting the required coordinates we used an Perl script .For finding the fasta sequence of the given coordinates we developed a script which would retrieve the sequences from various fasta files according to their location.
The target list were found to be in the following location
CHR2L
CHR2R
CHR3L
CHR3R
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CHRX
The fasta sequence of these genomic sequences were downloaded from the UCSC genome browser download page.
The next step of this project is to study the miRNA and retrieve sequence variation. For this a perl script is developed using Bioperl to blast the targets with the fastq sequences that are retrieved from the Drosophila genomic reference panel.This provides the FASTQ file for the entire genome of Drosophila. the divergence and variation of miRNA targets in the UTR region of duplicated genes, and offer insights into the role of these molecules in gene divergence can be studied by this method. These methods also helps us to identify miRNAs and their binding sites (i.e., targets) along with the availability of comprehensive genomic databases of SNPs provides an unprecedented opportunity to explore evolution at miRNAs and their targets.
References
Ambros, V., 2004. The functions of animal microRNAs. Nature, 431, 350-355.
Calin, G.A. & Croce, C.M., 2006. MicroRNA signatures in human cancers. Nat.Rev.Cancer., 6, 857-866.
Grun, D., Wang, Y.L., Langenberger, D., Gunsalus, K.C. and Rajewsky, N., 2005. microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput.Biol., 1, e13.
Lee, R.C., Feinbaum, R.L. and Ambros, V., 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75, 843-854.
Pegoraro, M. & Tauber, E., 2008. The role of microRNAs (miRNA) in circadian rhythmicity. J.Genet., 87, 505-511.
Reiter, L.T., Potocki, L., Chien, S., Gribskov, M. and Bier, E., 2001. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res., 11, 1114-1125.
Saunders, M.A., Liang, H. and Li, W.H., 2007. Human polymorphism at microRNAs and microRNA target sites. Proc.Natl.Acad.Sci.U.S.A., 104, 3300-3305.
Vasudevan, S., Tong, Y. and Steitz, J.A., 2007. Switching from repression to activation: microRNAs can up-regulate translation. Science, 318, 1931-1934.
Websites
http://hgdownload.cse.ucsc.edu/goldenPath/dm2/database/
