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The Search for Small Regulatory RNA Central Dogma: DNA to RNA to Protein hnRNA mRNA Replication Processing / Translocation rRNA tRNA Central Dogma: DNA to RNA to Protein hnRNA mRNA (unstable RNA) Replication Processing / Translocation rRNA tRNA (Stable RNA) Role of structure in stable RNA tRNA Tertiary structure Secondary structure G U A G U A Other stable RNA Telomerase 5' 3' DNA Replication RNA primer DNA polymerase DNA ligase 3' 5' 3' 5' Other stable RNA Telomerase DNA polymerase-independent repeated sequence Other stable RNA Telomerase Telomere growth Other stable RNA Spliceosome RNA hnRNA mRNA introns Other stable RNA RNAse P tRNA maturation Protein + RNA Other stable RNA SRP RNA Signal Recognition Particles protein RNA Other stable RNA • Fundamental processes of life (left over from RNA world?) • Nearly universal, highly conserved • Not many Basis of RNA function Sequence ??? Structure Function Prediction of RNA secondary structure • Energy minimization • Base-pairing energy • Stacking energy Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 1 with simple oligonucleotides Your name (no spaces) Enter anything you like as a login name, but no spaces or symbols. No password necessary. Click New Login General Syntax of BioBIKE Function-name Argument (object) Keyword object The basic unit of BioBIKE is the function box. It consists of the name of a function, perhaps one or more required arguments, and optional keywords and flags. A function may be thought of as a black box: you feed it information, it produces a product. Flag General Syntax of BioBIKE Function-name Argument (object) Keyword object Flag Function boxes contain the following elements: • Function-name (e.g. SEQUENCE-OF or LENGTH-OF) • Argument: Required, acted on by function • Keyword clause: Optional, more information • Flag: Optional, more (yes/no) information General Syntax of BioBIKE Function-name Argument (object) Keyword object Flag … and icons to help you work with functions: • Option icon: Brings up a menu of keywords and flags • Action icon: Brings up a menu enabling you to execute a function, copy and paste, information, get help, etc Clear/Delete icon: Removes information you entered or removes box entirely • Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 2 with mystery sequence 1 (a short oligonucleotide) Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 3 with mystery sequence 2 (a more realistic oligonucleotide) Conclusions re RNA structure prediction • A visually beautiful structure means nothing • A negative free energy means little • Function cannot be predicted from ability of sequence to form structure • Limited to 2-dimensional structure • Ignorant of pseudoknots • True structures can be predicted • Surrounding sequence can obscure true structure The world of RNA has changed Purple wild-type petunias Chalcone synthase tryptophan anthocyanin More enzyme, more purple? The world of RNA has changed Mutant petunias Cosupression Chalcone synthase Overexpression of exogenous chalcone synthase gene Overall expression of gene Basis of Cosuppression RNA virus Double-stranded RNA Basis of Cosuppression RNAInduced Silencing Complex siRNA Defense against foreign RNA Hannon GJ (2002) Nature 418:244-251 Small Noncoding RNA siRNA – Inhibits expression of exogenous genes RNAi – Human exploitation of phenomenon miRNA – Inhibits expression of endogenous genes miRNA expression Ambion Corp. http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html miRNA function Detection of transcribed RNA Genome mRNA Tiling microarray Shifts every 5 nt Covers 70 nt/spot mRNA Expect RNA found at regions of known genes Cheng et al (2005) Science 308:1149-1154 Detection of transcribed RNA Pooled results 10 human chromosomes, 8 cell lines Intergenic regions (31%) Many small ncRNA Known regions (different sources) Introns (26%) Cheng et al (2005) Science 308:1149-1154 Most RNA not attached to coding genes! …but Eubacteria? …but Eubacteria? Role of RNA-binding protein Hfq How to identify sRNA? • Label RNA with 32P; Isolate, characterize • Microarray detection • Shotgun clone by size class, sequence • Functional assay • Computational prediction How to identify sRNA? Sequence conservation Lenz et al (2004) Cell 118:69-82 How to identify sRNA? Structural conservation Lenz et al (2004) Cell 118:69-82 How to identify sRNA? Transcriptional features Promoter Short sequence Terminator How to identify sRNA? RNAz Combines two measures: Thermodynamic stability (Z score) Z= MFE – MFErandom σrandom MFE Minimum Free Energy How to identify sRNA? RNAz Combines two measures: Thermodynamic stability (Z score) Structural conservation Structural Conservation Index (SCI) • < 1 if structure poorly conserved • = 1 if perfect conservation • > 1 if structure perfectly conserved, with compensatory mutations The world's most populous organism Prochlorococcus A tiny marine cyanobacterium Strategy to find small RNAs Prochlorococcus MED4 Prochlorococcus #2 Prochlorococcus #3 Prochlorococcus #4 Conserved? Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 4, testing whether RNAz can find a known small RNA Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 5, testing whether RNAz predicts a small RNA between dmtB and trpD dmtB ??? trpD