For prediction of potential miRNA-mRNA interactions probably leading to mRNA degradation we have usedthe following four programs: TargetScan 5.1; miRDB; miRanda and Pictar.

TargetScan ( predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA1. Target sites that have imperfect seed matching, but contain compensatory 3’ sites are also predicted 2. In mammals, predictions are ranked based on the predicted efficacy of targeting as calculated using the context scores of the sites 3. The context score is the sum of the contribution of these four features: site-type contribution; 3' pairing contribution; local AU contribution; position contribution. TargetScanHuman considers matches to annotated human UTRs and their orthologs, as defined by UCSC whole-genome alignments.

miRDB (mirdb.org/miRDB) is an online database for miRNA target prediction and functional annotations in animals. All the targets were predicted by a bioinformatics tool called MirTarget2 4. Common features associated with miRNA target binding have been identified and used to predict miRNA targets. In this program the seed conservation is considered but not as a requirement. It includes seed composed from seed 8 (positions 1-8), positions 1-7 (seed 7a), positions 2-8 (seed 7b) and positions 2-7 (seed 6). Each seed type is scored differently. It also considers a composition of the regions next to the pairing site since a typical miRNA/target binding has a loop or bulge structure in this region. To formation of this structure lack of Cs is required. Presence and the position of pairing at 3’ miRNA site is also included in the analysis, as well as location in 3’UTR 5. The source of mRNA sequences is Genebank.

Pictar (pictar.mdc-berlin.de/) is design to favor perfect 7mer seed matches (either 1-7 or 2-8 nucleotides) at the 5’ site of miRNA. Imperfect seed matches are allowed, however much less scored than perfect matches. The results are then evaluated in respect of the free energy of the miRNA:mRNA duplex. Also conservation of the site is evaluated. Pictar takes into account the presence of multiple binding sites. The probability is assigned by cross-species comparison. The source of mRNA sequence for Pictar is UCSC and the miRNAs sequences are from Rfam 6.

miRanda ( analyzes the targets for miRNAs by the use of perfect seed matching. Wobbles are allowed but much less scored than perfect pairing, especially favored are pairing between 2-8 nucleotides. To the final score the presence of 3’ pairing is also incorporated as well as free energy of the duplex. Less conserved sites are excluded from prediction. UTR sequences were downloaded from UCSC genome browser7.

References

  1. Lewis BP, Burge CB, Bartel DP. Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets. Cell 2005; 120:15-20.
  2. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Research 2009; 19:92-105.
  3. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing. Molecular Cell 2007; 27:91-105.
  4. Wang X. miRDB: a microRNA target prediction and functional annotation database with a wiki interface. RNA 2008; 14:1012-1017.
  5. Wang X, El Naqa IM. Prediction of both conserved and nonconserved microRNA targets in animals. Bioinformatics 2008; 24:325-332.
  6. Krek A, Grün D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N. Combinatorial microRNA target predictions. Nat Genet 2005;37: 495-500.
  7. Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res 2008; 36: D149-153.