miRNABackground & Bioinformatics • Seungyoon Nam
RNA World Bioinformatics Seminar, By Yaron Levy
The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA" Craig C. Mello University of Massachusettsl Andrew Z. Fire Stanford University From Prof. Shin’s informal lecture at Stanford 2008.
miRNA : Discovery • lin-4 in C.elegans • first known to control the timing of C.elegans larval development. • does not code for a protein. • two species:small one 22 nt in length (mature miRNA), large one 61 nt in length (pre-miRNA). • antisense complementarity to multiple sites in the 3’ UTR of the lin-14. • reduces the amount of LIN-14 protein without noticeable change in levels of lin-14 mRNA.
microRNA • MicroRNAs are a family of small non-coding RNAs 18-22 in length that regulate gene expression in a sequence-specific manner. • microRNAs have been discovered in animals and plants, where the number of miRNAs is expected to 500-1000 per species. • Nearly all miRNAs are conserved in related species and many of them have homologs in distant species. • Also, microRNAs have been found in viruses such as Epstein-Barr virus, Kaposi sarcoma-associated herpesvirus and etc.
First miRNA in C.elegans miRNA in Plants miRNA in C.elegans with homologs In flies and human
miRNA : Biogenesis Posttranscriptional regulation Direct cleavage
Distribution of Mammalian miRNAs Found in Introns and Exons of Host Transcripts
miRNA: Function MiRNA mediated repression is affected by certain circustances. miR-122 miR-134
miRNA and Disease Blue bar: miRNA
Databases for miRNA From Prof. Shin’s informal lecture at Stanford 2008.
Computational identification of microRNA targets • Three major characteristic properties • MiRNAs are perfectly or near-perfectly complementary to their target mRNAs. • Although the complementarity between miRNAs and their targets were not perfect in animals, there still are some 5’ 7-9 nt seed regions between them. • The miRNA-mRNA duplex has a lower folding free energy. • Mature miRNAs, binding sites of mRNA to miRNA, and miRNA:mRNA duplex are highly conserved from species to species. Computational Biology and Chemistry 30,395–407 (2006)
Potent effect of target structure on microRNA function. For multiple sites in a UTR.
Potent effect of target structure on microRNA function. Only hybridization considered Real experiment Their result
The role of site accessibility in microRNA target recognition C ( genetically modified ~200bp construct to force it into highly paired (closed ) closed strucuter UTR N ( ~200 bp spanning miRNA target site and its neighborhood)
mirWIP: microRNA target predicition based on microRNA-contataing ribonucleoprotein-containing ribonucleoprotein-enriched transcripts Non-AIN-IP targets AIN-IP targets Comparison (seed pairing, accessibility, energy:S, A, E) Score system
mirWIP: microRNA target predicition based on microRNA-contataing ribonucleoprotein-containing ribonucleoprotein-enriched transcripts
Proteomics joins the search for microRNA targets-Proteome analysis Measuring target protein expression change using SILAC (Stable isotope labelling with amino acids in cell culture) http://en.wikipedia.org/wiki/Image:Silac.gif
Proteomics joins the search for microRNA targets-proteome analysis • mRNA expression change results in proteome expression decreased. • But a specific miRNA controlled proteome expression significantly by translation repression. • It implies translational repression has been underestimated in miRNA regulation.
Proteomics joins the search for microRNA targets-proteome analysis • Ectopic expression or overexpression of miRNAs might not produce the same effects as endogeneous miRNAs. • Blocking let-7b results in upregulated genes in Hela cells that would have down regulated on overexpression.