The Search for Small Regulatory RNA

2. The Search for Small Regulatory RNA

3. Central Dogma: DNA to RNA to Protein hnRNA mRNA Replication Processing /Translocation rRNA tRNA

4. Central Dogma: DNA to RNA to Protein (unstable RNA) mRNA hnRNA Replication Processing /Translocation rRNA tRNA (Stable RNA)

5. Role of structure in stable RNA A U G U A G U A G U A U A G U A G U A G G tRNA Tertiary structure Secondary structure

6. Other stable RNA Telomerase 5' 3' DNA Replication RNA primer DNA polymerase DNA ligase 3' 5' 3' 5'

7. Other stable RNA Telomerase DNA polymerase-independentrepeated sequence

8. Other stable RNA Telomerase Telomere growth

9. Other stable RNA Spliceosome RNA hnRNA mRNA introns

10. Other stable RNA RNAse P tRNA maturation Protein + RNA

11. Other stable RNA SRP RNA Signal Recognition Particles protein RNA

12. Other stable RNA • Fundamental processes of life (left over from RNA world?) • Nearly universal, highly conserved • Not many

13. Basis of RNA function Sequence Structure Function ???

14. Prediction of RNA secondary structure • Energy minimization • Base-pairing energy • Stacking energy

15. Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 1 with simple oligonucleotides

16. Your name (no spaces) Enter anything you like as a login name, but no spaces or symbols.No password necessary.Click New Login

17. Argument(object) Function-name Flag Keyword object General Syntax of BioBIKE 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.

18. Argument(object) Function-name Flag Keyword object General Syntax of BioBIKE 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

19. Argument(object) Function-name Flag Keyword object • 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 General Syntax of BioBIKE … and icons to help you work with functions:

20. Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 2 with mystery sequence 1(a short oligonucleotide)

21. 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)

22. 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

23. tryptophan anthocyanin The world of RNA has changed Purple wild-type petunias Chalcone synthase More enzyme, more purple?

24. Overall expression of gene The world of RNA has changed Mutant petunias Cosupression Chalcone synthase Overexpression of exogenous chalcone synthase gene

25. Basis of Cosuppression RNA virus Double-stranded RNA

26. Basis of Cosuppression RNA-InducedSilencingComplex siRNADefense against foreign RNA Hannon GJ (2002) Nature 418:244-251

27. Small Noncoding RNA siRNA – Inhibits expression of exogenous genes RNAi – Human exploitation of phenomenon miRNA – Inhibits expression of endogenous genes

28. miRNA expression Ambion Corp. http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html

29. miRNA function

30. mRNA mRNA Cheng et al (2005) Science 308:1149-1154 Detection of transcribed RNA Genome Expect RNA found at regions of known genes Tiling microarray Shifts every 5 ntCovers 70 nt/spot

31. Knownregions(differentsources) Detection of transcribed RNA Pooled results10 human chromosomes, 8 cell lines Intergenicregions(31%) Introns(26%) Most RNA not attached to coding genes! Many small ncRNA Cheng et al (2005) Science 308:1149-1154

32. …but Eubacteria?

33. …but Eubacteria?

34. Role of RNA-binding protein Hfq

35. How to identify sRNA? • Label RNA with 32P; Isolate, characterize • Microarray detection • Shotgun clone by size class, sequence • Functional assay • Computational prediction

36. How to identify sRNA? Sequence conservation Lenz et al (2004) Cell 118:69-82

37. How to identify sRNA? Structural conservation Lenz et al (2004) Cell 118:69-82

38. Promoter Short sequence Terminator How to identify sRNA? Transcriptional features

39. MFE – MFErandom σrandom MFEMinimum Free Energy Z = How to identify sRNA? RNAz Combines two measures: Thermodynamic stability (Z score)

40. 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

41. The world's most populous organism ProchlorococcusA tiny marine cyanobacterium

42. Prochlorococcus #2 Prochlorococcus #3 Prochlorococcus #4 Conserved? Strategy to find small RNAs Prochlorococcus MED4

43. 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

44. dmtB trpD 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 ???