Two short piecesMicroRNAWhat is microRNA?Slide 4Slide 5A model for miRNA functionHow to find miRNAs?Comparative genomicsPowerPoint PresentationFinding microRNA genesSlide 11Slide 12So what do we learn?Prediction Pipeline details: 1Pipeline details: 2Pipeline details: 3Final resultsSummary of part 1Part 2: finding the targetsPlant miRNAsSlide 21Near perfect complementarityAlternative Splicing (a review by Liliana Florea, 2006)What is alternative splicing?SignificanceSlide 26Slide 27Bioinformatics of Alt. splicingIdentification of splice variantsSlide 30Slide 31Slide 32Slide 33Splice variants from microarray dataSlide 35Identifying full lengh alt. spliced transcriptsMethod 1 (“gene indices”)Slide 38Problems with this methodMethod 2: Splice graphsSlide 41Splice graphsSummaryTwo short piecesMicroRNAAlternative splicingMicroRNA• First part is about discovery of the genes that code for microRNAs• Second part is about discovery of the “targets” of these microRNAsWhat is microRNA?•Genome has protein-coding genes•It also has genes that code for RNA–e.g., “transfer RNA” that is used in translation is coded by genes–e.g., “ribosomal RNA” that forms part of the structure of the ribosome, is also coded by genes•microRNAs are a family of small RNAs–genome has genes that code for microRNAs, i.e., the result of transcription is microRNAWhat is microRNA?•21-22 nucleotide non-coding RNA•The gene that codes for a miRNA first produces a ~70 nucleotide transcript•This “pre-miRNA” transcript has the capacity to form a stem-loop structure•This pre-miRNA is then processed into 21-22 nucleotide long miRNA by an enzyme called Dicer.What is microRNA?•Vast majority of microRNAs regulate other genes by binding to complementary sequences in the target gene•Perfect complementarity of binding leads to mRNA degradation of the target gene•Imperfect pairing inhibits translation of mRNA to protein•miRNAs are an important piece of the puzzle that is gene regulationA model for miRNA functiondoi:10.1016/S0092-8674(02)00863-2 Copyright 2002 Cell Press.How to find miRNAs?•Experimental methods so far•Lai et al (2003) one of the works that try solving this problem computationally•Basic idea:–look for evolutionarily conserved sequences–check if some of these fold well into the stem-loop structure (“hairpins”) associated with miRNAsComparative genomics•Start with 24 known Drosophila pre-miRNAs (the ~70-100 long transcripts before miRNAs)•All are found to be conserved beween D. melanogaster and D. pseudoobscura–Typically, more conserved than gene. (The third codon “wobble” not relevant here)miRNA genes are isolated, evolutionarily conserved genomic sequences that have the capacity to form extended stem-loop structures as RNA. Shown are VISTA plots of globally aligned sequence from D. melanogaster and D. pseudoobscura, in which the degree of conservation is represented by the height of the peak. This particular region contains a conserved sequence identified in this study that adopts a stem-loop structure characteristic of known miRNAs. Expression of this sequence was confirmed by northern analysis (Table 2), and it was subsequently determined to be the fly ortholog of mammalian mir-184. Most conserved sequences do not have the ability to form extended stem-loops, as evidenced by the fold adopted by the sequence in the neighboring peak.http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12844358Finding microRNA genes•Find highly conserved sequences, length ~70-100•Check for secondary structure•Are we done?–No, too many such sequences; more filters neededComparative genomics•Look carefully at pairwise alignments of each of the 24 pairs or orthologous pre-miRNAs.•Only three pairs completely conserved•Ten pairs are diverged exclusively within their loop sequence;no pair diverged exclusively in arm•Of the 11 remaining, seven show more changes in the loop than in non-miRNA-encoding armhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12844358So what do we learn?•That class 1 - 3 are the normal pattern of evolutionary divergence of miRNAs•That classes 4 - 6 are unlikely•Therefore use these criteria as additional filters for evolutionarily conserved sequencesPrediction Pipeline details: 1•Align the two genomes•“Regions” that should contain miRNA genes are estimated as those having–length 100, –<= 15% mismatches, –<= 13% gapsPipeline details: 2•Analyze conserved regions with mfold3.1, an RNA folding algorithm•Find the top scoring regions (from the mfold program) -- these are candidates for the next stagePipeline details: 3•Assess the divergence pattern of candidate miRNAs•Boolean filters: remove candidates with–exclusive divergence in arm–more divergence in miRNA-coding arm than in loopFinal results•200 candidate miRNAs came out•Experimental validation of many of these •24 novel miRNAs confirmedSummary of part 1•Learned what miRNAs are•and how the genes encoding these are predicted computationally•Learned that the miRNAs function to regulated gene expression by binding to the mRNA of the target genes (perfectly or imperfectly)Part 2: finding the targets•Rhoades et al (2002)•We should be looking for targets … •… with base complementarity•But small size (20-24 nt) and imperfect base pairing imply that we may ending up predicting too many•Rhoades et al found that nearly perfect complementarity is a good indicator of miRNA targets in plantPlant miRNAs•Started with 16 known Arabidopsis miRNAs•Looked for complementary strings with <= 4 mismatches and no gaps•Also did the same genome-wide search with “randomized” versions of the 16 miRNAsResults of this scandoi:10.1016/S0092-8674(02)00863-2 Copyright 2002 Cell Press.Near perfect complementarity•Number of hits with <= 3 mismatches is 30 for the real miRNAs, 0.2 for the random–Why fractional for random?•Therefore <= 3 matches supposed to be a good indicator of targets•Find all targets using this rule; as simple as that!Alternative Splicing(a review by Liliana Florea, 2006)What is alternative splicing?•The first result of transcription is “pre-mRNA”•This undergoes “splicing”, i.e., introns are excised out, and exons remain, to form mRNA•This splicing process may involve different combinations of exons, leading to different mRNAs, and different proteins•This is